JP2018113994A - Methods, substrates and systems useful for cell seeding of medical grafts - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods, cell growth substrates and devices that are useful in preparing cell-containing graft materials.SOLUTION: Described herein are methods, cell growth substrates and devices that are useful in preparing cell-containing graft materials for administration to patients. Tubular passages can be defined in cell growth substrates to promote distribution of cells into the substrates. Also described are methods and devices for preparing cell-seeded graft compositions, methods and devices for preconditioning cell growth substrates prior to application of cells, and cell-seeded grafts having novel substrates, and uses thereof.SELECTED DRAWING: Figure 10

Description

  This application was filed on May 25, 2010, which is incorporated herein by reference in its entirety, and is entitled “US Provisional Patent Application No. 61/348 entitled Methods, Substrates, and Systems Useful for Cell Seeding of Medical Implants. , Claim the profit of No.135.

  The present invention relates generally to medical materials and procedures, and in particular, to medical implants containing cells and methods, substrates and devices useful for their preparation.

  The field of regenerative medicine has shown great potential to improve the treatment of a wide variety of diseases or disorders in patients. One area of research is the field of studying implantable graft materials that contain living cells from patients or other sources. With respect to collection and reintroduction of patient-derived cells, so-called autograft cell therapy, methods, and systems are known for processing patient-derived tissue samples into cell preparations that can be reintroduced into the patient. It has been proposed that such methods and systems can be used "clinically" in a hospital setting. For example, in a single treatment in a hospital or in a single visit, a biological tissue sample is taken from a patient and processed into a cellular composition that is reintroduced into the patient.

  In one form of use, transplantable cell-containing grafts can be formed by combining cells to be introduced into a patient with a cell growth substrate. These uses may involve a culture period in which the number of cells increases after application to the cell growth substrate. Other uses do not involve such proliferation, and the cells are applied to the cell growth substrate and transplanted without a culture period.

  Although potential has been shown, the clinical realization of cell-containing graft materials has been delayed. There is a need for more convenient and / or effective methods or materials for binding cells to cell growth substrates so that they survive in the patient's body and often proliferate. The present invention addresses these needs in certain aspects of the present invention.

  The present invention, in some of its embodiments, provides methods, cell growth substrates, and devices useful for the preparation of cell-containing graft material for administration to a patient. The cell growth substrate of the present invention may include features that can enhance the binding between the substrate and the cell suspension. In certain embodiments, such features include a cylindrical passageway defined in the cell growth matrix that facilitates cell dispersion during administration of the cell suspension to the substrate. Further embodiments disclosed herein include a method and apparatus for preparing a flowable matrix graft composition seeded with cells, pre-treating the substrate prior to applying the cell composition to a cell growth substrate. Preparing an implantable implant in which cells are seeded by combining a method and apparatus for conditioning, a cell composition, and a matrix material that includes, for example, features for dispersing cells throughout the volume of the matrix material It relates to an automated method and apparatus, and to the preparation of a granular form of cell growth substrate and a fluid cell transplant material using the same.

In certain embodiments, a cell growth substrate is provided to support cell growth. The cell growth substrate includes at least one elongated cylindrical passage, the elongated tubular passage having a passage wall defining a lumen, the lumen being a first lumen on a surface of the cell growth substrate body. Extending from the opening into the interior region of the cell growth substrate. The lumen may be configured to receive a flow of the cell-containing liquid medium to disperse the cells within the internal region of the cell growth substrate. The cell growth substrate may be a cell growth matrix. The matrix may include collagen and / or may also include a synthetic polymer tubular element outside the matrix and fluidly bonded to the luminal opening. The substrate may include a plurality of cylindrical passages. The at least one tubular passage may include at least one primary tubular passage and at least one secondary tubular passage branched from the primary tubular passage. The matrix may include a remodelable collagenous extracellular matrix sheet material that retains growth factors, glycosaminoglycans, and / or proteoglycans from animal source tissue for the sheet material You may do it.

  In another embodiment, connecting a cell growth substrate, such as that described in the paragraph above, to a source of a liquid suspension of cells, the connecting step comprising transporting the elongated passageway to the source. A method is provided for preparing a cell seeded material comprising fluidly coupling to a lumen. The method also includes delivering the cells to an interior region of the substrate by transporting an amount of a liquid suspension of cells through the transport lumen and into the elongate passage. The connecting step includes inserting the substrate into a cell seeding chamber having an input tube fluidly connected to a source of a liquid suspension of cells, and fluidly coupling the input tube to the first lumen opening of the substrate. You may go out.

  In a further embodiment, a method is provided for preparing a composition seeded with cells for delivery to a patient. The method includes combining a fluid extracellular matrix particulate composition comprising extracellular matrix particles and a liquid medium with a liquid cell suspension to form a cell fluid composition. The method may also include mixing the cell fluid composition and / or gelling the cell fluid composition. This gelling step may include changing the pH of the cell fluid composition and / or changing the temperature of the cell fluid composition.

  Further embodiments provide a method for preparing and administering a cell graft. The method comprises the steps of preparing a preconditioned substrate comprising a serum protein composition and a biocompatible substrate by applying the serum protein composition to a biocompatible substrate suitable for administration to a patient. Including. A cell graft is prepared by adding cells to the preconditioned substrate and the cell graft is administered to the patient. Applying the serum protein composition may comprise applying the composition by spraying. The step of adding cells may include spraying the liquid cell suspension onto the preconditioned substrate and / or flowing the liquid cell suspension through the cylindrical passage of the substrate.

In another embodiment, an apparatus for seeding cells in a matrix is provided. The apparatus includes a first chamber for containing a liquid suspension of cells and a second chamber for containing a cell growth matrix material in which the cells are seeded. The seeding device further includes a passage for transferring an amount of a liquid suspension of cells from the first chamber to the second chamber, and a device for detecting at least one condition of the liquid suspension of cells. And an application device for applying an amount of a liquid suspension of cells to the matrix material contained in the second chamber. The application device may include at least one spray nozzle or at least one cannula having a lumen. The seeding device may also include a mechanism for moving the spray nozzle or cannula. When the application device includes a cannula, the mechanism for moving functions to retract the cannula proximally while dispersing the liquid cell suspension from the cannula opening, such as the distal-most opening. May be. The seeding device may also include an alignment structure for holding the matrix material in a predetermined position relative to the application device. A seeding device is also associated with the second chamber,
In addition, a dispersion assist device that functions to facilitate the dispersion of the cells in the matrix material after the cells are applied to the matrix material by the application device may be included. The dispersion assist device may function to generate a magnetic field in the matrix material, may be a mixer that functions to generate a flow in the liquid contained in the second chamber, and the second chamber May function to generate a pressure gradient within, may function to move the cell growth substrate contained in the second chamber, and / or rotate the second chamber to at least cause the cells to move. It may function to be dispersed in the cell growth matrix material in part by centrifugal force. Where there is a mixer, the mixer that functions to generate a flow may function to generate a pulsed bi-directional flow in the liquid contained in the second chamber.

  A further embodiment provides an apparatus for preparing a flowable, cell seeded implant. The device includes a first chamber containing a liquid suspension of cells and a flowable, cell-seeded graft material by combining the liquid suspension of cells with a flowable cell growth substrate material. A second chamber containing a flowable cell growth substrate material that is fluidly connected to the first chamber and into which the cells are seeded. The device also includes a mixer that functions to mix the flowable, cell-seeded graft material. The mixer may include a stationary mixer or a rotating mixer placed in the flowable, flowable, grafted material of the graft material. The first and / or second chamber may be a passage.

  In another embodiment, a method for preparing a cell-seeded substrate for treating a patient, including a human patient, and optionally a method for treating the patient are provided. The method includes processing a patient tissue sample to obtain a suspension of the patient's cells and placing a cell growth substrate in an incubation chamber of a cell seeding device. The method also includes initiating operation of the cell seeding device, wherein the operation detects at least one state of the cell suspension and binds the cells of the suspension to the cell growth substrate, The cells form a seeded substrate. In a method for treating a patient, the method also includes administering to the patient a substrate seeded with cells.

  Forming a gellable cell mixture by mixing the cells with a gellable composition having a first viscosity, and applying the gellable cell mixture to a cell growth substrate. Also provided is a method for preparing a seeded matrix. The method also includes gelling the gellable cell mixture to form a cell gel that contacts the cell growth substrate. The gel may have a second viscosity that is greater than the first viscosity. Gelation of the mixture may be caused by any suitable measure or combination thereof including, for example, changing (eg, raising or lowering) the pH of the mixture and / or changing (eg, raising or lowering) the temperature of the mixture. Also good. By contacting the mixture with the substrate, a layer can be provided on the outer surface of the substrate and / or the mixture is substantially homogeneous throughout the substrate (eg, when the substrate is porous) or at least a portion of the substrate. Can be dispersed. Subsequent gelation of the mixture provides an external cellized gel layer and / or the cellized gel is dispersed substantially homogeneously throughout or in part of the substrate.

  In a further embodiment, a cell seeded implant comprising an extracellular matrix matrix material, a collagenous gel applied to the matrix material, and cells attached to the matrix material, to the gel, or both, is provided. Provided. The collagenous gel may consist of an extracellular matrix hydrolyzate comprising natural collagen and at least one further natural bioactive substance derived from a source tissue for extracellular matrix hydrolysate. The at least one further natural bioactive substance may comprise growth factors, glycosaminoglycans, and / or proteoglycans.

  Further embodiments provide a method for preparing a cell graft for treating a patient, including a human patient, and optionally a method for treating the patient. The method includes collecting serum from a patient and obtaining a patient cell population by processing a patient tissue sample. The method further comprises preparing a serum-preconditioned matrix substrate by applying the serum to the matrix substrate, and forming a cell graft by applying the population of cells to the preconditioned matrix substrate. Steps. In a method for treating a patient, the method may also include administering a cell graft to the patient.

  In still other embodiments, methods of preparing materials for treating a patient and, optionally, methods for treating the patient are also provided. In this method, the cell suspension is combined with the granular cell growth substrate, and the cell suspension is incubated in contact with the granular cell growth substrate for a period and under conditions that do not significantly increase the number of cells. Forming a cellized granule having cells attached to the particulate cell growth matrix particles. In a method for treating a patient, the method may also include the step of administering the cellized particulate to the patient. In some forms, the incubating step of the method may be performed for a period and conditions such that at least 20% of the cells in the suspension adhere to the particles but do not increase the number of cells.

  Another embodiment provides a cell graft for treating a patient, and in some cases, manufacturing materials in a method for treating a patient with the cell graft and / or for treating a patient. The use of this cell graft in a method for providing is also provided. The cell graft includes a cellized granule composed of cells attached to extracellular matrix particles. When used for therapy, cell grafts may be introduced into the patient's body.

  Another embodiment provides a cell graft material comprising particulate cell growth matrix material and cells attached to particles of the cell growth matrix material. The cells include vascular endothelial progenitor cells, muscle-derived cells, or combinations thereof. The particles may be particles of naturally derived extracellular matrix sheet material that retains endogenous growth factors, glycosaminoglycans and / or proteoglycans by being separated from animal sources and optionally treated. The particles of such naturally derived extracellular matrix sheet material may be formed into regular shapes (eg, round, egg) by forming randomly generated particles by grinding the sheet material and / or cutting the sheet material. Molds and / or polygons) may be prepared.

  A further embodiment provides a cell graft that includes a filament made of extracellular matrix material and a group of cells attached to the filament. A filament may be a segment of a naturally derived extracellular matrix sheet material that retains endogenous growth factors, glycosaminoglycans, and / or proteoglycans by separation and processing from animal sources.

  Another embodiment provides a cell graft that includes a cell growth substrate in the form of a filament and a group of cells attached to the filament. The cells include vascular endothelial progenitor cells. The cell graft may be introduced into a patient's tissue in a further embodiment of the invention that provides a method for angiogenesis of cellular tissue.

Another embodiment provides a method for preparing a cell graft. The method comprises the steps of preparing a first cell growth substrate sheet and first forming a first cell seeding surface by applying a cell composition to the surface of the first cell growth substrate sheet. including. After the first application, the method includes laminating a second cell growth substrate sheet on the first cell seeding surface. The method may then also include applying the cell composition to the surface of the second cell growth substrate sheet.

  Another embodiment provides a cell graft that includes a first cell growth substrate sheet and a second cell growth substrate sheet laminated on the first sheet. The graft also includes a cell seeded layer between the first sheet and the second sheet.

  Another embodiment provides an apparatus for preparing a graft seeded with cells. The apparatus includes an incubation chamber for containing a cell growth substrate and an application device for applying a liquid cell suspension to the cell growth substrate, including a plurality of cannulas for supplying a volume of suspension. including. The device may be configured to retract the cannulas while delivering the amount of suspension from the cannulas.

  In yet another embodiment, a cell growth substrate composition is provided comprising a particulate material having sheet-form cell growth substrate particles having a small shape. In some forms, at least 25% of the sheet-form substrate particles have a first maximum cross-sectional dimension axis when considered in the plane of the sheet, the maximum cross-sectional dimension axis being perpendicular to this axis. In addition, it is not more than about twice the length of the second cross-sectional axis on the line centered on this axis. The maximum cross-sectional dimension of the substrate particles may range from about 20 microns to about 2000 microns. The matrix particles may be particles of naturally derived extracellular matrix sheet material that retains endogenous growth factors, glycosaminoglycans and / or proteoglycans by being separated from animal sources and optionally treated. Such naturally derived extracellular matrix sheet material particles may be prepared, for example, by cutting the sheet material to form particles of small shape (eg, circular, oval and / or polygonal).

  Another embodiment provides an extracellular matrix composition comprising particulate extracellular matrix material having sheet-form extracellular matrix particles having a small shape. At least 25% of the sheet-form extracellular matrix particles may have a first maximum cross-sectional dimension axis when considered in the plane of the sheet, the maximum cross-sectional dimension axis being perpendicular to this axis. Further, it may be less than about twice the length of the second cross-sectional axis on a line centered on this axis. The maximum cross-sectional dimension of the extracellular matrix particles may range from about 20 microns to about 2000 microns. The extracellular matrix particles may be particles of naturally derived extracellular matrix sheet material that retains endogenous growth factors, glycosaminoglycans and / or proteoglycans by being separated from animal sources and optionally processed. . Such naturally derived extracellular matrix sheet material particles may be prepared, for example, by cutting the sheet material to form particles of small shape (eg, circular, oval and / or polygonal). The extracellular matrix composition may also include cells attached to the particles, which in some embodiments cover substantially the entire outer surface of the particles and / or on the surface of the particles. A substantially confluent single layer is formed. The cell may comprise endothelial cells, vascular endothelial progenitor cells, or a mixture thereof and / or may be a clone. In some forms, the cells may consist of endothelial cells, vascular endothelial progenitor cells, or a mixture thereof.

  In another embodiment, there is provided a cell growth substrate material comprising a cell growth substrate material and an encapsulating material configured to encapsulate the cell growth substrate material and direct the flow of fluid medium through the substrate material. . This encapsulating material may define at least a first opening and at least a second opening spaced from the first opening.

Another embodiment provides a cell graft that includes a mixed cell population derived from adipose tissue and includes stem cells, vascular endothelial progenitor cells, leukocytes, endothelial cells, and vascular smooth muscle cells. The cell graft also includes a cell growth matrix consisting of extracellular matrix material and / or in the form of particles. The extracellular matrix material may include retained bioactive components derived from the source tissue for the extracellular matrix material. This retained bioactive component may be a growth factor, glycosaminoglycan and / or proteoglycan. When used, the granular cell growth substrate may contain sheet-form particles. For these purposes, sheet-form particles made of naturally derived extracellular matrix sheet material, for example, form particles of, for example, small shapes (eg, circular, oval and / or polygonal) by cutting the sheet material. May be prepared.

  In a further embodiment, a cell graft comprising a group of vascular endothelial progenitor cells and a granular cell growth substrate is provided. The particulate cell growth matrix may include sheet-form particles and / or may include extracellular matrix material. The extracellular matrix particles are naturally derived extracellular matrix sheet material that retains endogenous growth factors, glycosaminoglycans and / or proteoglycans by separating them from animal sources and optionally treating them for these purposes. The particles may also be Such naturally derived extracellular matrix sheet material particles may be prepared, for example, by cutting the sheet material to form particles of small shape (eg, circular, oval and / or polygonal), Or the fragment of the sheet material produced | generated by grind | pulverizing a sheet material at random, for example may be sufficient.

  Another embodiment provides a system for seeding cells in a matrix. The system includes a chamber for binding the cells to a cell growth substrate that is to be seeded with the cells. The system also includes a mechanism for assessing cell attachment to the substrate. This mechanism for evaluating may include a cell counter. A related matrix seeding method includes the steps of combining the cell growth substrate to be seeded with cells and the cells, and evaluating the attachment of the cells to the substrate. In a therapeutic method, the method may also include administering a substrate seeded with attached cells to a patient, including a human patient.

  Further embodiments of the invention and its features and advantages will become apparent from the further description herein.

FIG. 3 shows a side view of an embodiment of the cell growth substrate of the present invention. FIG. 3 shows a side view of another embodiment of the cell growth substrate of the present invention. FIG. 2 shows a top view of the substrate of FIG. FIG. 4 shows a top view of an alternative cell growth substrate of the present invention. FIG. 4 shows a top view of a further embodiment of a cell growth substrate. FIG. 6 is a top view of another embodiment of a cell growth substrate having a manifold supply. FIG. 4 shows a top view of another embodiment of a cell growth substrate. FIG. 6 shows a top view of another embodiment of a cell growth substrate having a large primary channel and a small secondary channel within the substrate. FIG. 2 shows a schematic diagram of an embodiment of a system for applying a cell composition to a cell growth substrate. FIG. 10 illustrates an embodiment of some secondary components of the system of FIG. Figure 10 shows another embodiment of some secondary components of the system of Figure 9; FIG. 3 shows a schematic diagram of another embodiment of an apparatus for binding a cell composition to a cell growth substrate. 2 shows a digital image of a small sheet particle of a decellularized ECM sheet whose composition is useful as a cell growth substrate. 2 illustrates a flowable cell graft material of the present invention associated with a delivery device. 2 shows a cellized filament graft of the present invention. Figure 3 shows a cellized filament graft of the present invention having a distal holding needle. FIG. 12B shows the graft of FIG. 12A housed in a delivery needle cannula when used for graft implantation. Fig. 2 shows a cell graft of the invention having a plurality of laminated cell growth substrate sheets. 1 shows a cell growth substrate that is a product of the present invention. 1 shows a cell growth substrate that is a product of the present invention. 1 shows a cell growth substrate that is a product of the present invention. Figure 3 shows a further cell graft structure of the present invention. Figure 3 shows a further cell graft structure of the present invention.

  In order to facilitate an understanding of the principles of the present invention, some reference will now be made to the embodiments illustrated in the drawings. These embodiments will be described using specific expressions. However, it will be understood that this is not intended to limit the scope of the invention. Any changes and further modifications in the described embodiments, as well as any further applications of the inventive principles described herein, will be considered to be normally envisioned by those skilled in the art to which the invention pertains. It is done.

  As disclosed so far, aspects of the invention provide for preparing a composition that can be used in some embodiments as a medical implant for a patient by combining cells with a matrix material. It relates to methods, materials and systems.

Cell Growth Substrate Having Defined Internal Passage Referring now to FIGS. 1-8, a variety of cell growth substrate structures that are particularly useful for combining with a cell composition to form a cell-containing graft material. Embodiments are shown.

  Specifically, FIG. 1 shows a side view of the matrix substrate 20. Substrate 20 includes a substrate body 21 preferably made of a porous matrix material suitable to support cell growth. The substrate body 21 includes a passage 22 defined therein, and this passage extends into an internal region of the substrate body 21. In the twentieth embodiment shown here, the substrate body 21 includes a first sheet 23 and a second sheet 24, and the sheet 23 defines a passage 22 between the sheet 23 and the sheet 24. 24. The sheet 23 and the sheet 24 are connected to each other along the connection area 25 in an area surrounding the passage 22. The connection region 25 may be formed by closely connecting the opposing surfaces of the sheet 23 and the sheet 24 by, for example, adhesion, fusion, gluing, cross-linking, stitching, or other methods. Accordingly, the cylindrical passage 22 includes a passage wall having a portion 26 defined by the surface of the sheet 23 and a portion 27 defined by the surface of the sheet 24 facing the same. In the embodiment shown here, regions 26 and 27 are generally rounded to provide passage 22 with a substantially circular or oval cross section. It will be appreciated that other configurations are suitable including passages having a polygonal or irregular cross-sectional shape. Connection region 25 has termination regions 28 and 29 in the lumen of passage 22. The termination regions 28 and 29 can thus occur as seams along the walls of the passage 22.

To prepare the substrate 20, one or more passage-forming elements are placed between the sheets 23 and 24, and then these sheets are connected to each other in the connection area 25, thereby providing these sheets. The one or more passage-forming elements may be sandwiched between them. As mentioned above, this connection may be by gluing, fusing, gluing or other methods. Where it is desirable for the passage 22 to be open at the outermost end of the substrate 20, the one or more passage-forming elements can reach the outermost ends of these sheets when the sheets 23 and 24 are overlaid or this outermost It may also provide the opening in the final product by extending beyond the end. In such a method of preparation, the one or more passage-forming elements are rods, rods, combs suitable for providing a passage to the finished product by maintaining a space between the sheets 23 and 24. It may be a structure, wire, tube, or any other element. In embodiments in which the one or more passage-forming elements are removed after the formation of the connection region 25, the outer surfaces of the passage-forming elements do not stick to or join to the opposing surfaces of the sheets 23 and 24. By being sufficient to do so, it is desirable to be able to remove the passage-forming element without damaging the passage 22 after formation of the connection region 25.

  In some embodiments, the passage-forming element or at least a portion thereof remains in the final product. Referring now to FIG. 2, one such embodiment is shown. The substrate 30 includes the same features as the matrix 20 and can typically be configured in the same manner. Thus, the substrate 30 includes a main body 31, a passage 32 to an internal region of the main body 31, and sheets 33 and 34 connected to each other. In addition, in the substrate 30, the tubular element 35 accommodated between the sheets 33 and 34 remains. The tubular element 35 may be made of a biocompatible polymer or other suitable material and may be left in the substrate 30 and implanted in the patient. The cylindrical element 35 may include a hardly degradable polymer (non-bioabsorbable) or a bioabsorbable polymer. In some embodiments, the tubular element 35 may include a bioabsorbable polymer that is completely absorbed after implantation into a patient. In the manufacture of the substrate 30, the cylindrical element 35 may be placed between the sheets 33 and 34 and these sheets then connected as described for the substrate 20 (FIG. 1). Alternatively, an element such as the substrate 20 may be first prepared and then the tubular element inserted into the passage 22. The tubular element 35 includes a porous wall, or a hole or perforation in the wall, so that the fluid cell suspension passes through the wall of the tubular element 35 and is in the region of the graft body 31 around it. It may be able to enter. In some forms, the tubular element 35 is opened by being less absorbent to the aqueous cell suspension than the material of the body 31 and / or by staying stiffer than the material of the body 31 when wet. The fluid is made up of the material that maintains the channel, thus allowing better flow of the aqueous cell suspension stream along channel 32 to allow this fluid to reach the inner region of substrate 31. By controlling the porosity or perforation of the tubular element 35, the wettability and the cell seeding of the substrate 31 when an aqueous cell suspension stream is introduced into the passage 32 can be optimized.

  Referring now to FIG. 3, a top view of the substrate element 20 of FIG. The passage 22 is indicated by an imaginary line (dotted line). The substrate 20 shown comprises a generally rectangular structure comprising a first pair of generally parallel sides 40 and 41 and a second pair of generally parallel sides 42 and 43 that are perpendicular thereto. It is. The passage 22 has an opening exposed at the side surface 40 of the substrate 20. The passage 22 in the illustrated embodiment is a blind hole and has a terminal end 44 located within the body 21 at a distance “d” from the side 41 of the body 21. In this way, a fluid composition, such as an aqueous cell suspension, can be passed from the side 40 into the passage 22, and this fluid composition is not carried by the passage 22 to the opposite side 41. Thereby, by creating fluid pressure in the passage 22, a cell suspension and / or another fluid, such as a substrate preconditioning medium, can be more quickly dispersed in the adjacent region of the body 21. . In some modes of use, pressure is applied to the blind hole passage 22 from the fluid introduced through the opening of the passage 22 in the side 40 to cause the fluid and lysed or suspended (eg, cellular) material to move into the body. It can flow into and through 21 volumes.

With reference to FIGS. 4-8, an alternate embodiment of a substrate element defining an internal passage is shown. Specifically, referring to FIG. 4, a cell having features similar to those of the substrate 20 of FIGS. 1 and 3 except that the passageway extends entirely from the first edge to the second edge of the substrate. A growth substrate 50 is shown. Specifically, the substrate 50 includes a first side surface 52, a second side surface 53 that is generally parallel to the side surface 52, a third side surface 54, and a fourth side that is generally parallel to the side surface 54. A generally rectangular body 51 having a side surface 55 is included. The substrate 50 includes a plurality of passages 22 that extend through the material of the body 51. The passage 22 has a first opening 56 group on the side surface 52 of the substrate 50 and a second opening 57 group on the side surface 53 of the substrate 50 opposite to the side surface 52. In this way, the passage 22 shows openings 56 and 57, which are spaced apart in the substrate body 51 and are in the opposite sides 52 and 53 in the embodiment shown. By flowing a flowable composition comprising cells through the passage 22 from the opening 56 to the opening 57, the cells can be implanted in the substrate 50 such that the cells pass through and contact the material of the body 51.

  Referring to FIG. 5, a cell growth substrate 60 is shown that is similar to the substrate 50 of FIG. 4 except that the first set of passages and the second set of passages intersect. Accordingly, the substrate 60 includes a substrate body 61 having a first side 62, a second side 63, a third side 64, and a fourth side 65. The substrate 60 includes a first plurality of passages 22 that have an opening 66 in the side surface 62 of the body 61 and an opposite opening 67 in the side surface 63 of the body 61. The substrate 60 includes a second plurality of passages 68 that pass through the body 61 in a direction transverse to the direction of the passage 22 and, in the illustrated embodiment, substantially perpendicular to the direction of the passage 22. It extends to. The passage 68 includes an opening 69 on the side surface 64 and an opposing opening 70 on the side surface 65. In some embodiments, the passage 68 intersects the passage 22 at passage intersections 71 that are fluidly coupled to the passage 22 and distributed throughout the body 61. In use, cells can be implanted in the substrate 60 by flowing the cell composition through passage 22 and passage 68. In this regard, the fluid cell composition may flow through passages 22 and 68 simultaneously or at different times, or they may flow in combination during the cell implantation operation. In embodiments in which the passages 22 and 68 intersect and are thus fluidly coupled to each other, turbulence, vortices, or passages 22 are also made at the intersection 71 by flowing a cell composition or other fluid through the passages 22 and 68 simultaneously. Fluid flow conditions can occur, such as a partial change in the actual flow along a vector that does not match 68. Thereby, the fluid can be easily removed from the passage and allowed to flow into the volume of the surrounding material constituting the substrate body 61. Thereby, cells or other substances can be more quickly or efficiently dispersed through the volume of the substrate body 61.

  Referring to FIG. 6, a cell growth substrate 80 having a plurality of passages 22 provided by a general manifold structure is shown. Specifically, the substrate 80 includes a main body 81 that has a plurality of passages 22 extending therethrough. A common supply passage 82 is fluidly coupled to the plurality of passages 22. The common supply passage 82 is fluidly coupled to an input passage 83 having an opening 84 with respect to the outside of the main body 81. Each passage 22 has an opening 85 to the outside of the main body 81. In use, a fluid, such as a cell composition, can flow through the opening 84 and into the input path 83 so that the fluid composition flows into the common passageway 82, which causes the fluid composition to enter the passageway 22. Flow through and disperse, and the fluid composition exits through opening 85. In this way, cells can be implanted into the substrate by circulating a fluid cell composition or other composition through the substrate 80 and possibly recirculating. As with the other embodiments described herein, this is accomplished by the passage 22 and possibly also the passages 82 and 83 being permeable to the composition, so that the composition is adjacent to the adjacent body 81. It only has to flow out into the volume.

Referring now to FIG. 7, another cell growth substrate 90 is shown. The substrate 90 includes a main body 91 and first, second, third and fourth side surfaces 92, 93, 94, 95. The body 91 includes a tortuous passageway 96 that extends through the material of the body 91 and has a first outer opening 97 and a second outer opening 98 that is spaced relative thereto. In the illustrated embodiment, the tortuous internal passage 96 includes a first bend 99 and a second bend 100 on the opposite side, with the bends 99 and 100 located in opposite directions. The generally linear passage portion 101 connects the bent portions 99 and 100 to each other. Thus, the winding passage 96 has a substantially sinusoidal shape that repeats from the opening 97 to the opening 98. Cells can be seeded into the volume of the substrate 91 by circulating a fluid containing cells from the opening 97 through the tortuous passage 96 to the opening 98 while allowing the cells and fluid to exit the passage 96. .

  Referring now to FIG. 8, a cell growth substrate 110 having a number of features in common with the substrate 90 is shown, and these features are numbered correspondingly. However, substrate 110 also includes a plurality of secondary passages 122 that are fluidly coupled to and extend out of primary passage 116. The diameter or cross-sectional area of the secondary passage is smaller than the winding passage 116. Accordingly, the secondary passage 122 can receive a flow of cellular fluid through the primary passage 116 and assist in the dispersion of the fluid and the cells associated with the fluid in the substrate body 111.

  In a further embodiment of the cell growth substrate shown in FIGS. 1 and 3-8, a separate cylindrical element is housed in some or all of the defined internal passages as described above with respect to FIG. You may be made to do. In addition, in a preferred embodiment, the sheets (eg, 23 and 24) used to prepare the indicated substrate are decellularized sheets of ECM tissue, preferably natural, as described below. (Intrinsic) It may be composed of a biologically active ingredient. Also, the substrate body shown in FIGS. 1-8 may consist of or consist of a porous sponge or foam matrix. This porous sponge or foam matrix can be formed, for example, by casting a matrix-forming material of any of those described herein around the passage, which is then A passageway can be provided by removing or leaving in the final product.

Cell / Substrate Treatment System and Method In a further aspect of the present invention, an apparatus for automatically seeding cells on a substrate is provided. FIG. 9 shows a schematic diagram of an embodiment of an automated cell seeding system 200. System 200 may optionally include an untreated tissue processing module 201 that processes an untreated or early stage tissue sample to provide a single cell suspension obtained from the tissue sample. The processing module 201 thus can serve as one or more functions common to this purpose, for example, by physical disruption of the tissue (eg, immersion softening), by tissue enzymes (eg, collagenase, trypsin) or other chemical cleavage, And / or may operate to perform functions including washing tissue or cellular components with saline or other suitable washing media. The tissue processing module 201 may also perform cell separation or enrichment steps, including, for example, immunochemical selection, fractionation, filtration, sedimentation or other similar operations for specific cell types or groups. Module 201 may also include, for example, growth substrate or growth medium and features for increasing cell number that provide suitable incubation conditions to increase cell number. Examples of systems for tissue processing that provide cell suspensions are US Patent Application Publication US 2005/0025755 published February 3, 2005, International Publication WO 2007/009036 published January 18, 2007. US patent application publication US 2008/0014181 published January 17, 2008, and US patent application publication US 2006/0141623 published July 29, 2006. Each of these publications is incorporated herein by reference in its entirety as teaching tissue processing methods and equipment for obtaining single cell suspensions suitable for use in module 201 of system 200. .

  The cell suspension provided from module 201 is transferred to cell suspension chamber 203 via conduit 202. A device 204 for detecting at least one state of the cell suspension optionally adjusts the parameters for applying the cell suspension to the substrate and / or for applying the cell suspension to the substrate. Provided to provide input for optimization. For example, the device 204 may be capable of functioning to determine the concentration of cells in the cell suspension in the chamber 203 and is functionally associated with the chamber 203. As a non-limiting example, the device 204 is a cell counting device, such as a Coulter counter, that obtains a cell count value based on the change in electrical resistance of the channel while the cell is passing through a liquid-filled channel. There may be. This change in resistance can be detected as a current or voltage pulse, which can be correlated to the concentration of cells in the cell suspension. The device 204 may also be a device that calculates a cell concentration value using light dispersion, such as forward light dispersion, using a laser. The device 204 may also be a device that determines the concentration of cells in a cell suspension using flow cytometry. Other means for measuring cell concentration will be apparent to those skilled in the art. It will also be appreciated that the particular means for measuring cell concentration is not critical to the systems and methods disclosed herein.

  To facilitate cell concentration analysis, the device 204 can withdraw a sample of cell suspension via a conduit 205 for analysis. Optionally, the device 204 may return this cell sample to the chamber 203 via the conduit 206 after analysis if the cell suspension in the chamber 203 does not cause undesirable contamination. If contamination occurs, the sample removed may be discarded via a disposal line (not shown). Apparatus 204 and / or chamber 203 may include valves and any pumps (not shown) suitable for moving cell samples through conduits 205, 206, as will be apparent to those skilled in the art. . Such valves and / or pumps may be controlled by the process controller 229, as described in more detail below.

  The system 200 may also include a processing media unit 250 that is fluidly coupled to the chamber 203 via a conduit 208. As with all other conduits for transferring fluid media within system 200, conduit 208 provides an optional valve (not shown) and power to transfer fluid from media unit 250 to chamber 203. It may be attached to the pump 209 that functions as follows. The pump 209 may be provided by a shared pump function in which one pump provides power to transfer fluid between some or all of the various chambers or other components of the system 200. Will be understood. Thus, although several separate pumps are described with respect to the system 200, these pump functions may be shared among the functions of the system 200 or may be a combination of them. It will be understood that there may be. Such valves and / or pumps may be controlled by a process controller 229, described in more detail below. The processing media unit 250 includes one or more chambers containing or filled with any of various media for processing or processing the cell suspension composition provided by the module 201. Also good. These media include, for example, washing media, cell culture media, or other materials to prepare the cells for application to the cell growth substrate, including, for example, different media for different stages of the cell growth phase. May be included.

The system 200 also includes an optimized cell prep chamber 210 in flow communication with the first cell suspension chamber 203 via a conduit 211, an optional valve (not shown), and an associated pump 212. Such valves and / or pumps 212 may be controlled by process controller 229 as described in more detail below. Thus, after some initial processing in chamber 203, the cell suspension composition may be transferred to chamber 210 for further processing by transferring it through conduit 211 by operation of pump 212. An apparatus 213 for determining the concentration of cells in the optimized cell preparation in chamber 210 is provided. The design of the device 213 may be similar to the device 204 described above. The conduit 214 may serve to transfer a sample of cell suspension composition from the chamber 210 to the device 213 for analysis. A conduit 215 may optionally be provided to return the sample to the chamber 210 if appropriate. Instead, as described above, the sample may be discarded through the disposal line after analysis.

Formulated media unit 216 is fluidly coupled to chamber 210 via conduit 217. Transfer of material from one or more chambers of unit 216 to chamber 210 may be facilitated by an optional valve (not shown) and pump 218. Such valves and / or pumps 218 may be controlled by process controller 229 as described in more detail below. Formulation media unit 216 may include a physiologically administrable material that can be administered to a patient in combination with a cell suspension. Formulation media can include, for example, saline, cell culture media, drugs such as antibiotics, substances designed to inhibit certain cellular reactions such as differentiation or cessation, or other materials. In some applications, the device 213 is used to determine the cell concentration of the cell suspension in the chamber 210. If the concentration of cells is higher than desired for providing to the cell growth substrate, a conditioned medium may be added from chamber 216 to dilute the cell suspension to obtain the desired cell concentration. The volume of the conditioned medium to be added may be calculated based on the total volume of the composition in the chamber 210 and the cell concentration determined by the device 213. The cell concentration values beneficial to the optimized cell preparation that are given to the cell growth substrate may range from about 10 ⁵ to about 10 ⁸ cells / ml, although other concentrations may be used. Alternatively, if the cell concentration is lower than the desired concentration, the chamber 210 may be functionally associated with a means for increasing the cell concentration, for example, by removing a certain amount of medium. In one embodiment, a filter 239 may be provided at the outlet 238 from the chamber 210, and the size of the filter hole is selected to allow cells to pass but not cell suspension fluid media. An optional valve (not shown) and pump 240 are provided to selectively remove a volume of fluid medium from the chamber 210 by pushing or pulling a volume of cell suspension against this filter. The cell suspension in the chamber 210 may be concentrated. If necessary, vibration may be applied to the filter by providing a vibration device 241 so that the filter is not clogged with cells during operation. In addition or alternatively, the filter may be flushed in reverse flow mode by configuring the system with appropriate pumps and / or valves to reverse the liquid flow through the filter. Such functions and associated values, pump 240 and / or vibration device 241 may be controlled by process controller 229 as described in more detail below. The material removed from the chamber 210 via the outlet 238 may be sent to a waste unit (“W”), for example. Optionally, the waste unit W is fluidly coupled to the chamber 203 or to other fluid sources in the system 200 to receive waste from this chamber or fluid source into one waste chamber or a separate waste chamber. Also good. After adjustments to increase or decrease the cell concentration in chamber 210 as a result of a concentration measurement initiated earlier, or as a result of introducing or removing media from chamber 210 otherwise, further It may be confirmed that the cell suspension is in a desired concentration range by removing the sample and placing it in the apparatus 213 for evaluation. A desired cell concentration may be obtained by further adjusting as necessary. After confirming that the cell suspension has the desired cell concentration, the cell suspension is powered from the chamber 210 to the applicator 221 by a pump 220 using an optional valve (not shown). Can be transported. Such valves and / or pumps 220 may be controlled by process controller 229 as described in more detail below.

  The application device 221 can be any of a variety of devices for applying the cell suspension composition to the cell growth substrate 223 contained within the seeding chamber 222. The applicator 221 may be stationary during operation, or may be movable so as to provide material to a larger area or volume that can be achieved if it is stationary during operation. The application device 221 may be controlled by the process controller 229 as described in more detail below. In certain embodiments, the application device 221 may be a spray nozzle or jet spray device similar to that found in spray devices, such as inkjet printers. Such spray devices are adapted to deliver the composition to the substrate by injecting the cell suspension composition against the substrate, either from a rest position or by moving in one, two, or three dimensions. What is necessary is just to arrange. In another embodiment, the application device 221 applies the cell composition to and / or in the surface of the cell growth substrate 223 while moving from a rest position or in one, two, or three dimensions. It may be a cannulated device having at least one cannula and preferably a plurality of cannulas. Some embodiments incorporate a cannulated applicator 223 having a cannula or multiple cannulas that are inserted into the substrate 223 while providing the cell composition, and the cell composition is, for example, a cannula that is a substrate 223. Is pushed out of the cannula while being inserted and withdrawn at a distal location within the volume of the cannula. In certain embodiments, such an application device includes a linear or other array of needles for applying a cell composition to the substrate 223. Still other modes of application will be apparent to those skilled in the art.

  The application device 221 desirably applies the cell composition substantially uniformly to the surface of the substrate or through the volume of the substrate. However, in other embodiments, the applicator 221 may be selectively seeded on certain portions of the substrate, so that the applicator 221 can apply other compositions of the substrate while applying the cell composition to a particular area of the substrate 223. The region may be configured to be free of cellular composition. Different types of applicators 221 may be combined into the system 200, for example, where the system includes both a spray applicator and a cannulated applicator.

In certain embodiments of the present invention, the system 200 is on a cell growth substrate that defines an internal passage for receiving a flow of cell composition, eg, a substrate as shown in FIGS. It has an application device 221 configured to seed cells therein. The application device 221 in such an embodiment desirably includes at least one cannula, preferably a plurality of cannulas. In certain embodiments, the cannula is fluidly coupled to a passage defined within the matrix (eg, passages 22, 68, 83, 84, 96, and / or 116 of FIGS. 1-8) to provide an amount of cellular composition. It functions to send things into this passage. In a further embodiment, a cannula is provided at each opening of the passageway to the outside of the cell growth matrix in the application device 221, where the cannula corresponds to and is aligned with such an opening. In the application device 221 that is stationary while applying the cell composition, the cannula may apply pressure to the cell composition into the passageway, whereby the cell composition flows through the passageway. , Exit from the side walls of the passage and enter the volume of the surrounding substrate material. For example, substrate passage 22 having spaced apertures 56 and 57 in FIG. 4, substrate passage 22 having spaced aperture pairs 66, 67 and 69, 70 in FIG. 5 and 68, substrate passages 83, 84 and 22 having spaced openings 84 and 85 in FIG. 6, and spaced pairs of openings 97, 98 and 117 in FIGS. In the case of a cell growth substrate that includes individual passages having openings spaced in the substrate, such as substrate passages 96 and 116 having 118 respectively, the cannula of the applicator 221 is fluidly connected to each opening. Also good. Such an application device 221 may function in, for example, a once-through mode, a counter-flow mode, or a combination thereof. In the flow-through mode, the input cannula pushes the cell composition into the first opening into the passage, and the discharge cannula passes through the passage and allows at least some of the cells of the input composition to enter the cell growth substrate. Receiving fluid after being placed in. In counterflow mode, the input cannula pushes the cell composition into both the first and second openings into the passage, and the input flow flows in the opposite direction in each passage. By creating pressure in the passageway in this way, a more uniform seeding operation can be performed simply by pushing the cell fluid out of the passageway into the surrounding volume of substrate material.

  In the flow-through mode, there may be only one passage for the cell composition through the substrate, but an advantageous embodiment is that the material received by the draining cannula is returned to the substrate passage at least once to further seed the cells. Will be implemented. This may, for example, provide a recirculation loop that collects the drained fluid and reverses the flow in the passage and / or directs the drained fluid back to the same input cannula and back to the drain cannula through the substrate passage. May be realized. In many embodiments, the original effluent fluid containing unseeded cells can be returned to the substrate passage multiple times to seed a higher percentage of the original cells of the cell composition onto the substrate.

  In this regard, refer to FIG. 9A in conjunction with FIG. 9, which shows one embodiment of a circulating loop application device 221 useful for seeding cell growth substrate 50 with a tube inside as shown in FIG. Is shown. The application device 221 has a plurality of input cannulas 221 a and a plurality of discharge cannulas 221 b, the input cannula 221 a is fluidly coupled to the opening 56 of the substrate 50, and the discharge cannula 221 b is fluidly coupled to the opening 57 of the substrate 50. . The input cannula 221a is provided in the input manifold 221c, which defines an internal input manifold passage 221d having an opening 221e, and the manifold passage 221d is fluidly coupled to each of the cannulas 221a to supply it. Opening 221e is fluidly connected to a conduit 219 (FIG. 9) that supplies the cell composition from chamber 210. The discharge cannula 221b is provided in a discharge manifold 221f that defines an internal discharge manifold passage 221g having a discharge opening 221h, and the discharge cannula 221b is fluidly coupled to the discharge manifold passage 221g to supply thereto. The circulation loop 221i is provided so as to fluidly connect the input opening 221e of the input manifold 221c and the discharge opening 221h of the discharge manifold 221f. A cell counter 221j, such as a counter that utilizes the Coulter principle, light scattering, or flow cytometry, may be fluidly coupled to the circulation loop 221i. As described above, the cell counter 221j can determine the cell concentration in the fluid circulating in the loop 221 in-line or by extracting and evaluating the sample. An optional valve (not shown) and a pump 221k are provided to encourage circulation in the application device 221 and sow the substrate 50. Such cell counters 221j, valves, and / or pumps 221k may be controlled by process controller 229 as described in more detail below.

  In use, the cell composition supplied from the conduit 219 to the circulation loop of the application device 221 continuously or intermittently in batch operation is used as the input manifold 221c, the input cannula 221a, the substrate 50, the discharge cannula 221b, and the discharge manifold. Cycle repeatedly through 221f and circulation loop 221i. During this operation, cells will be seeded on the substrate 50 and some cells will remain in the circulating fluid. Using the cell counter 221j, the cell concentration of the fluid circulating in the circulation loop 221i can be obtained continuously or periodically. The fact that an acceptable percentage of the initially given cells have been seeded on the substrate 50 means that when the cell concentration detected by the cell counter 221j reaches a desired or predetermined low value, the system 200 Determined and / or communicated to the user, for example, by using a process controller 229 described in more detail below. Thereafter, circulation within the application device 221 may be terminated and the substrate 50 may be prepared for administration to a patient immediately or after further incubation or incubation periods.

  As previously disclosed, in other embodiments, the application device 221 is configured to move while applying the cell composition to the substrate. Referring to FIG. 9B in conjunction with FIG. 9, a cell composition is provided to a cell growth substrate that is pre-fitted with a tube such as substrate 20 of FIG. 3 (shown by the dotted line in FIG. 9B) or substrate 50 of FIG. One such applicator 221 that is useful for is shown. The application device 221 of FIG. 9B includes a plurality of cannulas 221k attached to the input manifold base 221l. The input manifold base 221l defines an internal manifold passage 221m having an input opening 221n, and the manifold passage 221m is fluidly coupled to the cannula 221k to supply it. Input opening 221n is fluidly connected to conduit 219 of system 200, which supplies the cellular composition from chamber 210. The input manifold table 221l is mounted on a column 221o of the table, and this column is accommodated in a movable state at the opening of the column supply table 221p. An electric motor, solenoid, or other power mechanism (not shown) is mechanically coupled to the column 221o and preferably retracts the column 221o through the platform 221p at a constant speed or in another predetermined manner. This causes an action of pulling back the manifold base 221l and the cannula 221k associated therewith. The substrate 20, 50 is held in position during alignment with the clamping member 224a that engages at least a portion of the periphery of the substrate 20, 50 or with respect to the application device 221 during this pull back action. By other suitable mechanisms that are stably held in the initial position (see further description below regarding alignment device 224). The cannula 221k is initially housed in the passage 22 of the substrate 20 or 50, and its distal end is at a distal position within this passage, during which an automated pullback operation is initiated during which the cell The composition is pumped at a predetermined rate and extruded from the tip of cannula 221k. In this way, by controlling the pullback rate and flow rate from the cannula 221k, the system 200 can very effectively seed a cell growth matrix with a given fluid cell composition.

  With continued reference to FIG. 9, as described above, the system 200 holds the cell growth substrate 223 in a predetermined position relative to the initial or in-process position of the application device 221 or in the chamber 222. An alignment device 224 may be included. In this way, the device 221 may function reliably to implant the cell composition onto the substrate 223 in the desired manner. The alignment device 224 may include, for example, upstanding walls, clamps, pins, or other features that hold the substrate 223 in place within the chamber 222 in conjunction with the material or shape of the substrate 223. Further, the substrate 223 may comprise mechanical elements that cooperate with the alignment device 224 while being fed into the chamber 222. For example, the substrate 223 has a rigid structure of the alignment device 224 by having a relatively rigid plastic strip or bar that is temporarily attached (non-implantable) or permanently attached (implantable). And may be stably arranged in the chamber 222 in cooperation with each other. After sowing and prior to implantation, such mechanical elements may be removed from the substrate 223 if not intended to be implanted or suitable for implantation.

The system 200 may also include a dispersion assist device 225 to facilitate dispersing cells throughout or through the substrate 223. Accordingly, the dispersion assist device 225 may function to apply force to the cell composition in the chamber 222 and / or on the substrate 223. The dispersion assist device 225 may function to more evenly distribute the cells in the substrate 223, for example, by creating a vacuum and pulling the cell composition and thus the cells through the thickness of the substrate 223. Alternatively, the dispersion support device 225 may be a magnet such as a permanent magnet or an electromagnet, and may provide a magnetic field surrounding the substrate 223. This, together with the magnetic material associated with the cell, for example, magnetizes the liposome to which the cell attaches and may be used to disperse the cell through and / or along the substrate 223. . Alternatively, the dispersion support device 225 may function to support the dispersion of the cell composition in the substrate 223 by applying a motion such as vibration or rotation to the substrate 223. In some embodiments, the dispersion assist device 225 may move the cell composition with centripetal force by providing a high speed rotation, and the substrate 223 may move into the path of the cell composition to be moved (eg, substantially in the case of rotation in a horizontal plane). (Along vertical or upright walls). Using this mechanism, the cell composition can be moved relative to the substrate 223 into the substrate 223. For example, a device 225 that can provide a pulsatile and / or bidirectional flow to the liquid in the chamber 222 to enhance these two connections by applying force to the cell composition and / or substrate 223. Other devices 225 may be used. In an embodiment, the sheet-like substrate 223 may be placed in the chamber 222 to divide the chamber 222 into first and second volumes, the first and second volumes being mutually exclusive except for portions of the substrate. Fluid sealed. Next, a directional flow is created in the chamber 222 by creating a pressure difference with the aid of the dispersing device 225, thereby creating a transmural flow of cell suspension in the substrate 223, which causes the cells to move into the substrate. Disperse over 223, optionally in substrate 223. All functions of the chamber 222 may be controlled by the process controller 229, described in more detail below.

  In other embodiments, an extracellular matrix hydrolyzate or gel material, including, for example, those described herein, is placed in the chamber 222 with the cells, and a solid, sterically stable cell growth substrate is added to the hydrolyzate / You may incubate in the mixture of a gel and a cell, including this, but being completely immersed in it. The combined material may be agitated as described herein. The solid, sterically stable substrate may be an ECM material as described herein that preferably retains at least one natural (endogenous) bioactive component or a combination thereof as described herein. And / or sheet, filament, thread, or granule. The bound material may be administered to the patient after incubation as described herein.

  In still other embodiments, the applicator 221 may have one or more cannulas as described herein, wherein the cannula discharge opening is a sponge, foam or other similar fibrous substrate. Etc. may be placed in the volume of the solid cell growth substrate 223. The cell fluid may then be injected into the internal volume of the substrate material 223, so that the cell fluid undergoes pressure and moves toward the outer surface of the substrate 223 (possibly outside the outer surface of the substrate), On the way, the cells are planted in the substrate. In certain embodiments, the cannula and its / their openings and the substrate 223 are configured such that fluid material flows substantially uniformly in all directions from a generally central location within the substrate throughout the substrate 223. The cells may be easily dispersed uniformly within the substrate 223.

During and / or after seeding and incubating for the substrate 223 described herein, the system 200 evaluates the seeded substrate 223 so that a significant number of unattached cells are It may be determined whether or not it remains on or in 223. This can be done, for example, by forcing a non-adherent or poorly attached cell to flow by flowing a liquid inspection pulse through all or part of the substrate 223, and then collecting this liquid pulse to You may carry out by evaluating the presence or absence. This assessment includes a quantitative assessment in some embodiments. A test pulse may be sent, for example, from a chamber in media unit 226 (described further below) to chamber 222 via line 227, or another independent source and supply line may be provided. The test pulse solution is, for example, physiological saline, preconditioning medium, cell culture medium, or other liquid that allows detection of unattached or poorly attached cells on the substrate 223 after migration. There may be. In certain embodiments, as described herein, collected pulse volumes with displaced cells may be sent to the removed cells to detector 213, which may, for example, employ Coulter principles. It may be a cell counting device used or another means of counting cells. If an excessive number of cells are moved by the test pulse, the cells may adhere by incubating the substrate 223 and the cells for an additional period. If there are no cells collected in the test pulse or if the number of collected cells is small enough, the seeded substrate 223 may be removed from the chamber 222 and administered to the patient. The system 200 may optionally generate a signal, such as a visual signal and / or an audible signal, to indicate that the seeded substrate 223 in the chamber 222 is ready for administration to a patient. All of these functions may follow the instructions of the process controller 229, further described below.

  System 200 may also include a preconditioning media unit 226 having one or more chambers fluidly coupled to application device 221 via conduit 227 and powered from pump 228. The application device 221 may share the role of providing the cell composition and preconditioning medium to the substrate 223, or another application device may be incorporated. The preconditioning unit 226 may include and supply a medium for preconditioning the substrate 223 to condition to receive the cell composition. The preconditioning medium in chamber 226 may be, for example, a cell culture medium or may contain proteins or other substances that are beneficial to the cell or that make the substrate 223 more suitable for cell survival. In certain embodiments, the preconditioning medium in unit 226 includes serum that receives cell grafts generated using system 200, preferably autologous serum from the patient. The preconditioning medium may be provided to the substrate 223 using the application device 221. Optionally, the preconditioning unit 226 or other component of the system 200 may incorporate material for rinsing the substrate 223 after processing with other preconditioning media for substrate testing or other work. Also good. For these purposes, in some embodiments, the chamber 222 may include a drain connected to a waste line.

  System 200 is preferably automated and thus includes a process controller 229, which includes pumps, valves, temperature control units, detectors such as devices 204 and 213, application device 221, dispersion assist device 225, and others of system 200 By controlling various mechanisms within the system 200, such as the components of, the operations described herein can be implemented. Process controller 229 may be a processor-based system for controlling all of the various features of system 200 either fully automatically or with the aid of user control. Although not shown for clarity in FIG. 9, the process controller 229 communicates with various mechanisms of the system 200 that the controller 229 controls or provides sensed data to the controller 229 via appropriate input and / or output communication paths. Combined. This will be apparent to those skilled in the art.

  Process controller 229 may include an electronic user input device 230 such as a keyboard and / or mouse connected via connection 231. A display 232 connected via connection 233 may be provided to display status entries, analysis results, attention to the user, and other information as desired. Provide printer device 242 connected via connection 243 to print analysis results, current status of cell seeding work, and / or record of work completed by system 200 during cell seeding work, and as desired Other information may be generated. These devices are provided with user control instructions to the process controller 229 so that the system 200 operates as necessary for the current cell seeding operation, and various forms of information are displayed, printed, or user Combined to be manipulated by.

The process controller 229 is a personal computer, a workstation computer, a laptop computer, a palmtop computer, a tablet computer, a wireless terminal having a calculation function (for example, a mobile phone equipped with a Windows (registered trademark) CE, a palm operating system, or portable information). Terminal (PDA)) or a microcontroller embedded in system 200. It will be apparent to those skilled in the art that other computer system architectures can be employed and that the particular architecture chosen is not critical to the systems and methods disclosed herein.

  Generally, such a process controller 229, when implemented using a computer, is a bus for communicating information, a processor coupled to the bus for processing information, and an information and processor coupled to the bus. Main memory for storing instructions for and a read-only memory coupled to this bus for storing static information and instructions for the processor. A display device 232 is coupled to the bus for displaying information to a user of the system 200, and an input device 230 is coupled to the bus for communicating information and user command selections to the processor. A mass storage device interface for communicating with a data storage device containing digital information may also be included in the process controller 229 along with a network interface for communicating with a network.

Processors include PENTIUM®, CORE® and XEON® microprocessors manufactured by Intel Corporation, POWER PC or POWER ISA manufactured by IBM Corporation (IBM Corporation), Sun Corporation ( It can be any of a wide range of general purpose processors or microprocessors, such as SPARC processors manufactured by Sun Corporation. However, it will be apparent to those skilled in the art that various other processors can be used in any particular computer system. Display device 232 may be a liquid crystal device (LCD), a cathode ray tube (CRT), a plasma monitor, a light emitting diode (LEC) device, or any other suitable display device. The mass storage device interface allows the processor to access the digital information in the data storage device via the bus. The mass storage device interface may be a universal serial bus (USB) interface, an integrated drive electronics (IDE) interface, a serial advanced technology attachment (SATA) interface, etc., coupled to the bus for transmitting information and instructions. . Data storage devices include conventional hard disk drives, floppy disk drives, flash devices (such as jump drives or SD cards), compact disk (CD) drives, digital multipurpose disk (DVD) drives, HD DVD drives, Blu-ray ( An optical drive such as a registered trademark DVD drive, or another magnetic, solid state, or optical data storage device may accompany the associated media (floppy disk, CD-ROM, DVD, etc.).

  Generally, a processor uses a mass storage device interface to retrieve processing instructions and data from a data storage device and downloads this information to random access memory for execution. The processor then executes the instruction stream from random access memory or read only memory. The instruction selection and information input by the input device 230 are used to guide the instruction flow executed by the processor. Next, various mechanisms in the system 200 may be controlled using the processing execution result.

  Process controller 229 is configured to generate output for display on display device 232 and / or for driving printer 242 to print a hard copy. Preferably, the display device 232 is also a graphical user interface for allowing the user to interact with the displayed information.

The process controller 229 may also be configured to communicate with one or more external systems (not shown) via a network such as a local area network (LAN), a wide area network (WAN), or the Internet. Both the process controller 229 and the external system may be configured to function as a web server, as a client, or as a web server and client, and may be enabled by a browser. As such, system 200 may remotely access and / or store information, may be remotely controlled and / or monitored, and exchanges data between process controller 229 and other systems. It may be done.

  In order to minimize losses during cell transfer operations, it may be desirable to use as few chambers as possible in processing cell suspensions. Thus, in one embodiment, the system 200 is not provided with an optimized cell prep chamber 210 and associated detector 213, but instead provides a cell suspension to the substrate in the first chamber 203. It may be processed so as to be in a desired state. Thus, in this alternative embodiment, a conduit 234, shown in dotted lines, fluidly connects chamber 203 to application device 221. The conduit 234 may receive power from the pump 235 to transfer the optimized cell suspension to the device 221. This more simplified type of system 200 may also include a conduit 236 that feeds from the conditioned media chamber 216 to the cell suspension chamber 203 and is powered by a pump 218. Thus, the cell suspension composition may all be processed in one chamber 203 using the processing medium from chamber 250 and the conditioned medium from chamber 216. For subsequent processing steps using various media, chamber 203 may be a means for removing a given media such as the filter or liquid after processing media is provided from chamber 250 and / or 216. Other devices that can remove or increase the concentration of cells may be provided. After processing and adjusting the cell suspension composition in the chamber 203, the device 204 may be used to confirm that the cell concentration of the cell suspension composition is within the desired range; This composition may be sent to the application device 221 for application to the substrate 223. For ease of handling and operation, the system 200 may include a housing 237 that houses some or all of the components described herein.

Referring to FIG. 10, a schematic diagram of another automated cell seeding system 250 is shown. The system 250 may be used to prepare a flowable cell graft, which in certain embodiments is by a minimally invasive technique such as a penetrating needle, catheter, or other percutaneous delivery device. May be delivered. System 250 includes many components in common with system 200 (FIG. 9) described above, which are numbered the same in FIG. 10 and need not be repeated. With respect to other components, the system 250 is a cell for containing a particulate form substrate, a gel form substrate or precursor material thereof, or combinations thereof, optionally with other materials for the implant. A growth substrate input chamber 251 is included. The treated cell suspension composition is powered from pump 220 using an optional valve (not shown) and binds to cell growth substrate in chamber 251 via conduit 219. The prepared composition, including cells and matrix material, is transferred from chamber 251 to vessel 254 by flow through conduit 252 powered by pump 253 using an optional valve (not shown). Such valves and / or pumps 220 and 253 may be controlled by process controller 229. The container 254 may be a chamber device that is temporarily attached to the conduit 252 and fluidly connected, and may be removed to carry the cell graft material from the system 250. Container 254 may thus be a vial, bag, or other container that is fluidly connected to conduit 252. In certain embodiments, the container 254 may include a delivery device for delivering the cell graft composition or components thereof to the patient. In one embodiment, the container 254 includes a chamber, such as a syringe barrel, from which the cell graft composition is forcibly evacuated by a flow-coupled needle, catheter or other minimally invasive device to the patient. Sent to the organization. System 250 includes a structural engagement feature for inserting and engaging container 254 such that such container 254 can receive fluidized graft material that is fluidly coupled to conduit 252. You may go out.

  Chamber 251 may include a device 255 for mixing materials within chamber 251. The device 255 may consist of a mechanism that is located entirely outside the chamber 251 but provides motion to the chamber 251 or its components to mix the components therein. For example, such devices may include rotating elements such as vibrators, stirrers, vortex generators, or static mixers located in the flow path for the components to be mixed. The device 255 may also consist of a mechanism located within the chamber 251 such as a paddle, stir bar or impeller. Such a mechanism is driven by a motor or other means to stir the flowable composition contained in chamber 251. The device 255 is also comprised of both internal and external elements of the chamber 251, such as an external moving (eg rotating) magnet that drives a magnetically coupled paddle, a stir bar or other element within the chamber 251. It may be. Device 255 may be controlled by process controller 229.

  In addition to providing a mixing function within chamber 251, system 250 may include a thermal controller 256 associated with chamber 251 and / or a thermal controller 257 associated with vessel 254, which are It functions to control the temperature of each material in the chamber and container. Thermal controllers 256, 257 may be controlled by process controller 229 and may include appropriate temperature sensing elements that provide temperature feedback measurements to facilitate temperature management. The thermal control device 256 may be a heating element and / or a cooling element. In one mode, the thermal controller 256 may function to manage its viscosity by controlling the temperature of the material in the chamber 251. As an example, the cell growth substrate composition contained in chamber 251 may include a gelable material, and thermal controller 256 may provide a cell suspension in which gelable material is supplied from conduit 219. The material may be functioned to remain ungelled before and during bonding with the liquid. In this way, the lower viscosity of the matrix material will further facilitate mixing with the cell suspension material. In this application, for a gellable material that increases in viscosity with decreasing temperature, the thermal controller 256 functions to heat the material in the chamber 251. In this regard, it is desirable to perform the heating so that the cells are not significantly damaged by the heat, such as heating to a temperature below about 42 ° C. when the cells are present. In certain embodiments, a gelable material within the substrate composition is selected that gels at 37 ° C. or slightly above. In order to mix with the cell suspension to obtain a substantially homogeneous composition, the gellable material is brought to a temperature above its gel point but below that which causes significant damage to the cells ( For example, it may be heated at less than about 42 ° C. After mixing, the cell / matrix composition may be maintained at a temperature above the gel temperature of the matrix material, for example using thermal controller 257, before and during administration to the patient. In this manner, the material is placed in a lower viscosity (and more fluid) delivery state while being delivered, for example, using a needle or other cannula, but the patient (eg, about 37 ° of normal heat). After being delivered to a human patient at 0 ° C., it gels and increases viscosity accordingly. This makes it easier to maintain the delivered graft material at the site of administration. Alternatively, the cell / matrix material gels or gels outside of the patient (eg, by cooling with devices 256 and / or 257) after being mixed and substantially homogenized. May be delivered as a higher viscosity implant.

For a gellable substrate material that gels with increasing temperature or otherwise increases in viscosity, the thermal controller 256 may function to cool the material in the chamber 251. In this regard, it is desirable to perform cooling so that the cells are not significantly damaged by freezing, such as cooling to a temperature higher than about 0 ° C. In certain embodiments, the gelable material in the substrate composition may or may not gel at a temperature of about 37 ° C., for a lower temperature, eg, a temperature generally below room temperature (about 25 ° C.). If this is the case, select a material that increases viscosity. In order to obtain a substantially homogeneous composition by mixing with the cell suspension, the material may be cooled using the device 256 to maintain a low viscosity state. After mixing, the cell / matrix composition may be kept cool using thermal control devices 256 and / or 257 before and during delivery. In this way, the material has been made more fluid while being delivered, for example through a needle or other cannula, but has been delivered to a patient (eg, a human patient having a normal temperature of about 37 ° C.). Later, the viscosity increases, making it easier to maintain the delivered graft material at the site of administration. Alternatively, after mixing to a substantially homogenous state, the cell / matrix material is warmed using, for example, apparatus 256 and / or 257, or warmed at the ambient setting, viscosity May be delivered as a higher implant.

  Conditions other than temperature may be used to gel the cell growth substrate material when desired (eg, after mixing with cells). For example, a collagenous material that gels with an increase in pH may be selected. During the mixing operation in chamber 251, the pH of the substrate / cell composition may be kept at a relatively low level that allows the cells to survive, at least during the mixing period, while the fluidity of the composition remains higher. By way of example, such pH may be in the range of about 4 to about 6.5. After mixing, the collagenous material may be gelled to increase the viscosity of the graft material by increasing the pH of the composition. Such pH adjustment may be performed by adding a biocompatible base or buffer or both. These additives may be provided from a chamber in unit 226 or from another reagent chamber provided in system 250. The pH of the composition after mixing may be in the range of about 6.5 to about 8, for example. Desirably, the pH after mixing is suitable for administration to a patient and in certain embodiments from about 7 to about 7.5.

  Immediately after the cell growth matrix material is combined with the cell composition in the system 200 or 250 as described above, the seeded graft material may be administered to the patient immediately. In other embodiments, the graft material seeded with cells may be incubated outside the body for a period of at least sufficient to allow at least a portion of the cells to adhere to the cell growth substrate. The cells that adhere to the cell growth substrate are desirably a substantial proportion of cells, for example, a proportion of cells that is greater than about 20% of the cells initially provided. The duration of the incubation phase for attaching such cells is from 1 minute to about 5 hours, and in certain embodiments from about 5 minutes to about 3 hours. During this period, the composition attaches to the surface by at least some of the cells in the composition due to its natural nature and adheres to the cell growth substrate sheet, particles or other materials in the combined composition. Kept in state. While the cell attachment phase or cycle is taking place, the number of cells initially given may not increase significantly, for example, the percentage of viable cells in this composition will No more than 10% of the cells bound to the substrate, and in certain embodiments, the number of cells of the composition is substantially the same or less than the number of cells originally bound to the cell growth substrate. Few.

  In preparing the flowable graft material in the automated system 250, the composition may optionally be stirred continuously or periodically during the cell attachment phase, eg, using a mixer device 255. . In certain embodiments, the composition is agitated gently and periodically using a mixer device 255 during a cell attachment cycle. A relatively quiet cell attachment phase occurs between the agitation phase. For example, each of the plurality of non-stirred phase periods may be about 3 minutes to about 20 minutes, with a shorter stirrer phase being sandwiched between, for example, about 10 seconds to 5 minutes. This agitation is performed gently so that the suspended free cells are further brought into contact with the cell growth substrate particles or material but no significant migration of cells already attached to the substrate material occurs.

  During and / or after seeding and incubating for a period of time in the chamber 251 described herein, the system 250 can evaluate the flowable substrate cell / substrate mixture to determine whether it is not attached to the substrate. It may be determined whether a number of cells remain in the suspension medium of the mixture. This may be done, for example, by collecting the sample and filtering to remove the substrate material to which the cells have adhered, leaving only cells that are freely suspended in the suspension medium. This suspended free cell is then sent to detector 213 or another independent detector, utilizing the Coulter principle, light scatter or other means for counting cells as described herein. And count. In another mode, the entire medium from chamber 251 (including, for example, particulate substrate, suspension medium and cells) may be sampled and inspected using the Coulter principle, light scattering or other means. Evaluation values for the medium (eg, scattering or electrical resistance) vary depending on the percentage of cells attached to the freely suspended cells (eg, to the substrate particles), so the evaluation value is acceptable for cell attachment to the substrate. It may be associated with a level. If too many cells remain free in the suspension medium, the cells and cells may be allowed to attach by incubating the substrate and the cells for an additional period of time. If there are no cells left free in the suspension medium or if the number of such cells is very small, the seeded substrate material may be removed from chamber 251 and administered to the patient. Similar to system 200, system 250 optionally generates signals such as visual and / or audible signals (eg, on display 232) so that the seeded substrate material in chamber 251 can be administered to a patient. May be shown. All of these functions may follow the instructions of the process controller 229.

  In the systems 200 and 250 disclosed above, the chambers described may be provided by any suitable configuration including, for example, bags, vials, passages, plastic containers, and the like. The conduits described may be provided by tubing, a lumen through the larger plastic structure of the system, or any other suitable configuration. It will also be appreciated that by providing a valve in the chamber and / or conduit, the flow can be selectively turned on and off depending on the situation in conjunction with a pump or other material transfer means. Those skilled in the art will readily be able to conceive of these and other physical system features given the disclosure herein.

  The systems 200 and 250 may be configured such that the residence time of the cell graft material prior to implantation into the patient is relatively short, for example up to about 3 hours. Such a configuration will typically be designed to attach cells to the substrate without significantly increasing the number of cells (eg, the number of cells does not increase or the increase is less than 10%). . However, in certain embodiments, the systems 200 and 250 are configured for longer incubation and culture periods of cell graft material so that the number of cells is equal to the number of cells initially seeded on the substrate. For example, an increase greater than 20% may be shown, for example, and some embodiments may exhibit an increase greater than 100%. For these purposes, the systems 200 and 250 may also include a mechanism for applying a force, such as shear force, strain, or tension, to the substrate during the culture period. These forces can affect the growth and fractionation of the seeded cells during the incubation / culture period, resulting in a different protein expression pattern from the same cells when incubated / cultured without force. Indicated. In this way, the cell graft can be improved for a given end use in the patient's body.

In certain embodiments, system 200 or 250 may also include a secondary cell incubation chamber that is independent of chamber 222. By culturing cells different from those incubated in the chamber 222 in the secondary chamber, signal molecules such as hormones, cytokines, growth factors, etc. transferred to the chamber 222 may be secreted. . This signal molecule can, for example, regulate the growth or differentiation of the cell by contacting the cell during culture in the chamber 222. Transfer of signal molecules from the secondary incubation chamber to the chamber 222 may occur, for example, by pumping these molecules through a conduit, by flow through a membrane, or by other means. In some forms, the signal molecule will be effective to send a higher percentage of stem or progenitor cells cultured in chamber 222 to a given differentiation pathway. Control of the secondary cell incubation chamber may be performed by the process controller 229 or optionally by another process controller in communication with the process controller 229.

Particulate multicell / matrix graft material In one embodiment of the present invention, provided flowable cell graft material comprises multicellular bodies suspended in a liquid medium, each multicellular body comprising a cell. It consists of cell growth matrix particles to which is attached. The maximum cross-sectional dimension of the substrate particles may be from about 20 microns to about 2000 microns, and in some embodiments from about 100 microns to about 1000 microns. The substrate particles may be substantially uniform in size with respect to each other, for example, having a maximum cross-sectional dimension within about 20% or 10% of each other, or may be different in size from each other ( For example, there is a relatively small particle and a large particle, and in some cases, a controlled overall mass by mixing two or more substantially uniform particle groups among different particle groups. Forming a group of particles). In an advantageous variant, the substrate particles are in sheet form, the thickness of the sheet may be from about 20 to about 2000 microns, more preferably from about 20 to about 500 microns, and / or the surface of the sheet (e.g. high The maximum cross-sectional axial length when considered in terms of width or width is in the range of about 25 to about 2500 microns, more preferably in the range of about 100 to about 1000 microns, greater than the thickness of the sheet. The thickness of the sheet, in certain embodiments, may be from about 20 to about 1000 microns, and / or the maximum cross-sectional axis length when considered in the plane of the sheet ranges from about 100 to about 1500 microns. It may be. In addition or alternatively, the substrate particles may be relatively round and small rather than long and fibrous when considered in terms of the sheet. The shape of the substrate particles may be regular to each other or irregular to each other. In certain embodiments, the particles may be sheet-like particles that are generally circular, oval and / or polygonal (eg, having 3 to 10 sides, such as a triangle, square or rectangle, pentagon, hexagon, etc.). . For example, the maximum cross-sectional dimension axis of the substrate particles, or a substantial proportion (eg, greater than about 25%) of the substrate particles in the composition when considered in terms of sheet, is perpendicular to the maximum cross-sectional dimension axis. No more than about twice the length of the cross-sectional dimension axis on the line centered on this maximum cross-sectional dimension axis, preferably at least about 50% of the substrate particles have this feature, more preferably at least about 70% Substrate particles have this feature. Such particulate cell growth matrix materials also constitute embodiments of the present invention used alone (eg, as a cell-free tissue graft material) or in combination with the cell compositions described herein.

The small sheet-like substrate particles may be cut out from a larger substrate material sheet. In certain embodiments, the larger matrix material sheet is an extracellular matrix sheet taken from a tissue source and decellularized as described herein. Sheet-like particles having the above characteristics may be cut from larger ECM sheets using a mechanical tool such as a punch or die, or by cutting with a laser, or using other suitable means. In the desired embodiment, the cutting method used is that the natural bioactive ECM feature or natural bioactive ECM molecule is in the larger starting ECM sheet material being processed, as described in more detail herein. In some cases, this feature or molecule is not excluded. In addition, the ECM sheet being processed, and the resulting ECM sheet particles, may have a natural epithelial basement membrane retained on one or both sides of the sheet material and / or one side of the sheet or It may have a basement membrane component deposited by biosynthesis on both sides. In order to provide a natural epithelial basement membrane on both sides of the sheet, two separate decellularized ECM layers, each having one basement membrane side and the opposite side, are stacked, with the basement membrane side facing outward What is necessary is just to melt | fuse or adhere | attach each other. Next, the resulting sheet composed of two layers may be processed to form the sheet-like particles. To prepare particles with a non-natural basement membrane component deposited, the decellularized ECM sheet is treated by growing the outer skin, endothelium, or other cells on both sides to deposit the basement membrane component. May be. Next, the cells may be removed while leaving the basement membrane component, and then the sheet-like particles can be prepared by treating the sheet.

FIG. 11 shows a digital image of a very small exemplary ECM “dot” that was laser cut from a larger ECM sheet. The ECM sheet utilized in this particular example is Cook Biotech, West Lafayette, Indiana.
A monolayer of porcine small intestine submucosa available from Incorporated. It can be seen that the length of the maximum cross-sectional axis in the plane of the sheet is about 505 microns, and the length of the cross-sectional axis perpendicular to and centered on this maximum cross-sectional axis is about 413 microns. Thus, these two length dimensions are within about 25% of each other, resulting in a small sheet particle structure. This degree of correlation in the length dimension of particles greater than about 50% of the ECM particle composition is particularly beneficial in use, especially the maximum cross-section in the plane of the sheet for particles greater than about 50%. Useful when the axis range is from about 100 to about 1000 microns.

  In order to prepare a graft composition seeded with cells, the granular growth substrate may be combined with a cell preparation using, for example, the system 250. For flowable grafts, the granular growth matrix may be suspended in a liquid medium such as an aqueous medium. Prior to administration, the graft composition may be incubated for a cell attachment cycle such as any of those described above. The size and shape of the granular cell growth matrix is relatively flat and small, resulting in favorable suspension and cell attachment properties, which can be improved with flexible matrix materials such as extracellular matrix sheet materials It can also be made. For administration to a patient, a graft seeded with fluid cells may be placed in a syringe or other delivery device to deliver the graft to the target tissue for transplantation. As an example, with reference to FIG. 11, a medical device 300 is shown that includes a flowable cell graft composition 301 contained within a syringe 302. The cell graft composition 301 includes a plurality of cellized bodies 303 including matrix particles 304 as described herein and in the foregoing description, with a group of cells 305 attached to each matrix particle 304. In certain embodiments, the cells 305 form a generally confluent layer that covers the matrix particles 304. The cellized body 303 is suspended in a liquid medium 306, such as an aqueous medium that optionally contains nutrients for the cells and is physiologically compatible with humans or other patients. Cell graft composition 301 is fluid and is contained within barrel 307 of syringe 302. Plunger 308 is contained in barrel 307 and is operated linearly to push composition 301 out of its opening 310 through a flow-coupled needle 309. As such, the medical device 300 can be used to administer the composition 301 into the patient's tissue. In certain preferred embodiments, the target tissue is in need of revascularization, and the cell graft body 303 is, for example, an endothelial cell or vascular endothelial progenitor cell comprising endothelial colony forming cells as described herein in certain embodiments. It includes cells 305 capable of forming blood vessels. Matrix particles 304 assist in retention of cells 305 in the target area when injected into the target tissue. In particularly preferred embodiments, the particles 304 are extracellular matrix particles as described herein.

Cell graft with matrix matrix coated with gel The cell graft of the present invention may have a porous matrix cell growth matrix that is at least partially coated with a gel, such as an extracellular matrix gel. The cells in this case are in the gel, on the surface of the gel, or both. The gel may be applied to the porous matrix substrate before mixing with the cells, after mixing with the cells, during mixing with the cells, or some combination of these. The gel may include substances that enhance the property of cells to adhere to the substrate, such as fibronectin, laminin, collagen I, or other materials. The gel or gel precursor material, when given mixed with cells, may be easier to apply to the substrate by giving it in a low viscosity (eg, not gelled), and if desired, cells And may penetrate into the porous network of the substrate. The gel or gelable precursor is then allowed to gel, or it is gelled or its viscosity is increased, prompting the gel and the cells therein to immediately attach to the porous matrix substrate. May be. In one embodiment, the gel is a U.S. patent application published on Apr. 12, 2007, U.S. Patent Application Publication No. US2007082060, U.S. Patent Application on Aug. 25, 2004, which is incorporated herein by reference in its entirety. It consists of the extracellular matrix gel described in application No. 10 / 569,218. Thus, the gel can comprise an ECM hydrolyzate composition prepared by acid and / or enzymatic degradation of ECM tissue as described herein. The composition can gel when the pH is increased to about 6.8 to about 8 and / or the temperature of the material is increased to about 37 ° C. Such compositions include the natural collagen and natural (endogenous) bioactive non-collagen component of the starting ECM material, such as growth factors, glucosaminoglycans, proteoglycans, and / or other materials described below with respect to ECM materials. obtain.

  The system 200 may be used to prepare these cell grafts having a matrix substrate coated with a gel. For example, a gel or gel precursor may be applied to the substrate 223 using the application device 221. A gel or gel precursor may be supplied from the preconditioning media unit 226 and applied to the substrate 223 to apply the gel material before the cells. By combining cells and gel material in-line with those provided from conduits 219 and 227 prior to release from application device 221 to bind the cells to the gel or gel precursor prior to application to the substrate. , Mix. Alternatively, the cellular material may be combined with the gel or gel precursor in the chamber of the preconditioning media unit 226 or in another chamber prior to application to the application device 221. If necessary, temperature and / or pH control can be performed by the system 220 to easily increase the viscosity of the applied material after contact with the substrate 223. As an example, to adjust the pH, a sufficient amount of basic material, such as NaOH, is combined with the cell / gel precursor composition just prior to application to the substrate 223 to sufficiently delay the complete gelation of the material. May be applied to the substrate 223 and then stabilized. Alternatively, substrate 223 can be preconditioned with a basic or buffer material to neutralize or increase the pH of the more acidic cell / gel precursor material when contacted with substrate 223. May be. Further, the temperature of the cell / gel precursor material may be raised by a heating element before and / or after application to the substrate 223. These and other adjustments to form the gel may be automated by the system 200.

Cell Graft Filament In a further embodiment, the present invention provides a cell graft comprising a cell growth substrate in the form of an elongated filament, with a group of cells attached along the filament. In these embodiments, the filament length may be at least about 1 mm, for example, in some embodiments in the range of about 1 mm to about 30 mm or about 5 mm to about 30 mm. The maximum cross-sectional dimension of the filament may also be from about 20 microns to about 2 mm, or from about 100 microns to about 1 mm. For these purposes, the extracellular matrix substrates described herein are preferred. To prepare such cell grafts, the elongated substrate is placed in the presence of a cell suspension containing the desired cells for at least a sufficient period of time for the cells to adhere to the substrate, eg, as described herein. Incubation may be in an automated system, such as the described system 200 or system 250. Referring to FIG. 12, several cellized filament grafts are schematically shown. Each of the cellized filament grafts 230 includes an elongated cell growth substrate 321 and a group of cells 322 attached to the substrate 321. The cells 322 may form a generally confluent layer that covers the cell growth substrate 321 in certain embodiments. To obtain this, sufficient cells initially applied may adhere to the substrate 321 to form a layer, or after seeding on the substrate filament 320, this is generally sufficient to form a confluent layer. You may culture as much as possible. In use, the cellized filament grafts 320 are individually placed, for example, each celled filament graft 320 longitudinally into the needle lumen, the needle inserted into the desired target tissue, and the graft 320 may be introduced into the treatment site by pushing it out of the needle using fluid pressure. This causes the implant 320 to disperse the cells 322 through the elongated region of the target tissue. This may be useful, for example, when the development of one or more blood vessels in the area is desired. For these purposes, angiogenic cells such as endothelial cells or vascular endothelial progenitor cells including the ECFC cells may be used. In an alternative embodiment, a flowable cell graft suspension comprising a plurality of elongated filament grafts 320 may be placed in a syringe and injected into the target tissue. Such a suspension and a medical product for its delivery are shown in FIG. 11 except that an elongated graft 320 is used instead of or in addition to the relatively smaller granular cell graft body 303. This is the same as the product 300 to be manufactured.

  Referring now to FIG. 12A, another cellized filament graft 320A of the present invention is shown. The graft 320A may have the same characteristics as the graft 320, including an elongated filament matrix 321A and a group of cells 322A attached to the matrix. The implant 320A further includes a fixation element that engages the patient's tissue, which in the illustrated embodiment has a shaft portion 324A and a barb end 325A that engages the tissue, Shown as hook 323A. The barbs 323A or other fixation elements are attached to or near the end of the filament substrate 321A in this exemplary embodiment. This attachment may be done by tying, by welding, by gluing, by integral formation, or by other suitable means. The anchoring element, such as the barbs 323A in one embodiment, resists passage in one direction in tissue more strongly than passage in the opposite direction. This may be achieved, for example, by directional barbs 325A. The barbs 323A or other anchoring element may be made of a permanent material or a bioabsorbable material. As an example, the persistent or bioabsorbable material can be formed of a metal or polymer material. Suitable bioabsorbable polymers include glycolic acid polymers, lactic acid polymers, or copolymers of glycolic acid and lactic acid, polycaprolactone, and other known materials. In use, the barbs 323A or other fixation elements resist movement of the implant 320A once the implant 320A has been implanted into tissue such as the patient's muscle or other tissue.

Referring now to FIG. 12B, in some modes of use, the implant 320A may be combined with a delivery cannula 326A, such as a needle cannula. In one form, all or a portion of the filament matrix 321A of the implant 320A may be housed in the lumen 328A of the cannula 326A. Similarly, the barb element 323A may be partly or wholly housed in the cannula 326A, or the barb 323A may be provided, for example, distally and completely outside the cannula 326A. In the illustrated embodiment, the shaft 324A or at least a portion thereof is received in the lumen of the distal region of the cannula 326A, and the barb 325A is distal end 327A (eg, a needle) threaded through the tissue of the cannula 326A. It is located beyond the tip of Using the combined device in this state, by extruding cannula 326A and barbs 325A into the patient's tissue receiving graft 320A to the desired depth of tissue under the patient's skin "S". It may be inserted. Thereafter, a force is applied to retract cannula 326A and retract cannula 326A by retracting cannula 326A while barb tip 325A engages and resists the patient's tissue. While delivering from lumen 328A. Implant 320A is then implanted directly into the patient,
Cell population 322A may grow in treatment of a patient in certain embodiments. In certain variations of this invention, cell population 322A includes endothelial cells and / or vascular endothelial progenitor cells, including, for example, endothelial colonies that form the cells described herein. The tissue to be treated and transplanted with the graft 320A may be a tissue requiring angiogenesis, for example, ischemic tissue of the myocardium or ischemic tissue caused by severe limb ischemia. . In such applications, transplantation of a group of cells 322A into an elongated region along the substrate 321A can generate one or more blood vessels along the elongated implantation region. However, other types of cell populations 322A and other diseases, defects, or illnesses can be treated using the filament graft 320A.

Cell Graft Having Laminated Substrate Layer The cell graft of the present invention may comprise a plurality of cell growth substrate sheets in a laminated configuration, with a group of cells between the laminated sheets. In some cases, the outermost layer of this structure is also formed. In order to prepare such a graft, cells may be seeded on the first cell growth substrate layer, for example, by applying a liquid film containing a cell suspension to at least one side of the sheet. Next, a second substrate layer is stacked on the side of the first substrate layer on which the liquid film is provided, and then another cell liquid film is applied to the exposed side of the second substrate layer. That's fine. If desired, the process may be repeated to form a structure composed of laminated substrates, with cells evenly or locally distributed throughout the thickness of the structure. FIG. 13 shows a schematic diagram of such a laminate graft structure 330. The structure 330 includes a plurality of stacked cell growth substrate layers 331, preferably a harvested and purified extracellular matrix tissue sheet as described herein. These stacked layers 331 may partially or completely overlap. Cell layers 332 each consisting of a group of cells 333 are provided between the sheets 331 and optionally also on the outermost surface of the structure 330. Prior to implantation, the structure 330 may be incubated for at least sufficient time for the cells to adhere to the sheet 331, which may contribute to the stability of the structure as an integrated graft unit. In an alternative example, the first seeded cell population is grown by incubating the structure 330 during the culture period. Also, prior to applying cells 333 to sheet 331 during construction of structure 330, sheet 331 may be preconditioned with blood components such as serum or serum proteins and / or other culture media components such as nutrients, salts, and the like. Also good.

  Laminated cell graft structures such as structure 330 may be prepared in an automated system such as system 200 of FIG. For this purpose, the first substrate sheet 331 may be placed in the chamber 222 and the treated cell suspension may be applied to the upper surface of the sheet 331 using the application device 221. Next, the second substrate sheet 331 may be placed on the first sheet, and an additional amount of cell suspension may be applied to the second sheet 331 using the application device 221. This process may be repeated multiple times, for example 2, 3, 4, or 5 times. In addition, preconditioning media may be supplied from the media unit 226 and applied to each sheet 331 prior to providing the cell suspension. The first, second, and subsequent sheets may be sequentially placed in the chamber 222, either manually by the user or using an automatic feeding mechanism provided by the system 200.

  FIG. 17 shows another embodiment of a cell graft of the present invention having a laminated growth substrate sheet. The cell graft 380 comprises a first cell growth substrate sheet 381 and a cell material 382 laminated on the surface of the sheet 381, preferably any of the extracellular matrix layers identified herein. Including. Cell material 382 may be any of the cells identified herein, flowable cell growth material or substrate, eg, granular cell growth substrate as described herein, and / or gel cell growth as described herein. Can be included with the matrix material. After the material 382 is deposited on the surface of the sheet 381, a second cell growth substrate sheet 383 is laminated on the material 382 to create a laminated or sandwiched cell graft.

  FIG. 18 shows yet another embodiment of the cell graft of the present invention. Graft 390 includes a first cell growth substrate sheet 391 having a plurality of wells 392 defined therein and extending only in a portion of the thickness of sheet 391. Cellular material 393 is deposited in the well 392. Cell material 393 may be, for example, simply a group of cells, or, as described further below, the cells may be supplied with a flowable cell growth material such as a particulate substrate or a gel-formed substrate or a combination thereof. It may be a combination. A second cell growth substrate sheet 394 is laminated over the first sheet 391 to cover the filled well 392 and optionally at least temporarily confine the cellular material 391 within the well 392. The cell growth substrate sheets 391 and / or 394 and some embodiments are any of the ECM layers described herein.

Cell growth substrate product having a flow directing layer The present invention also provides a product in which the cell growth substrate material is covered or wrapped with a second material. The second material is less permeable to fluids such as aqueous cell compositions than the cell growth substrate material. The covering or wrapping material can facilitate cell distribution in the material by facilitating the flow of cell fluid through the length or thickness of the cell growth substrate material. Referring to FIG. 14, a cell growth substrate product comprising a cell growth substrate material 341, such as any of the materials described herein, and a permeable or semi-permeable encapsulant 342 surrounding the cell substrate material 341. 340 is shown. The encapsulating material 342 may define a first opening 343 and a second opening 344 that is spaced from the first opening 343. In certain embodiments, the openings 343 and 344 are each on opposite sides of the cell growth substrate material 341. Further, in alternative embodiments, multiple openings 343 and / or 344 may be provided. In use, the product 340 is connected to a source of liquid cell suspension, and the suspension is flowed into the opening 343 by pressure, thereby allowing the cell suspension material to flow through the cell growth substrate 341 and causing the cells to flow through the substrate 341. Or cells may be embedded in the substrate 341. The cell suspension fluid may exit through openings 344 with at least some of the original cells lost. Optionally, fluid exiting opening 344 may be recirculated through opening 343 to seed at least some remaining cells on substrate 341. Cell growth substrate material 341 may be monolithic, granular, or other cell growth substrate material as described herein. The encapsulating material 342 may be implantable to the patient, or may be removed prior to transplantation of the cell growth substrate 341 after cell seeding, which is not intended for transplantation. The encapsulating material 342 can be, for example, a natural or synthetic polymeric material that is permanent or bioabsorbable upon implantation. The material 342 may be encapsulated using a bioabsorbable synthetic polymer such as that disclosed elsewhere herein.

Referring to FIG. 15, another cell growth substrate product of the present invention is shown. The product 350 includes a cell growth substrate 351, such as any of the substrates disclosed herein, and a first encapsulant 352 that contains the material 351. The permeability of the encapsulating material 352 to the liquid in which the cells are suspended or from which the cells are suspended, such as water or another aqueous medium, is lower than that of the substrate material 351, for example in water or other aqueous medium. Suspended or will be suspended. The encapsulating material 352 may be, for example, a plastic or other polymer tray in which the material 351 is accommodated. This tray or other encapsulating material 352 may define a first opening 353 and a second opening 354 spaced from the first opening 353. A second encapsulant material 355 covers and seals the opening defined by the tray or other material 352. The encapsulating material 355 may be, for example, a polymer film that can be peeled from a tray or other encapsulating material 352. For these purposes, the film or other material 355 may include an exposed and grippable portion 356, desirably at the periphery thereof, to grab the material 355 into the tray or other material 352. When used to peel from, an opening defined by a tray or other material 352 may be opened and exposed for removal of the cell growth substrate 351. Thus, during a cell seeding operation as described above in connection with FIG. 14, the material 355 retains a seal against the material 352 and thus confines the cell growth substrate 351, thereby allowing fluid to flow from the opening 353 to the opening 354. To help the cells seed through the volume of material 351. After this seeding process, the encapsulating material 355 may be peeled from the material 352 and the seeded substrate 351 may be removed and implanted into the patient.

  Referring now to FIG. 16, another embodiment of a cell growth substrate product 360 is shown. Since the product 360 is similar to the product 350 in many respects, the product 360 has the same numbered features except that it is numbered starting with “360”. Product 360 further includes a fluid distribution function, at least in fluid inlet opening 363, preferably associated with both this opening and fluid outlet opening 364. Desirably, these functions facilitate substantially plug flow of fluid through the product 360. The cross-sectional dimension of this “plug” is substantially the cross-sectional shape of the substrate 361. This will aid in uniform distribution of the cells through the substrate 361. For these purposes, the tray or other encapsulant 362 defines branch walls 367 and 368 that branch off from the opening 363 toward the periphery of the cell growth substrate 361. Desirably, the ends of the inner surfaces of the branch walls 367 and 368 are at two points substantially on the outer periphery of the cell growth substrate 361. Thus, after the fluid entering from the opening 363 fills the gap 369 defined on the proximal side of the substrate 361, the pressure of the fluid entering from the opening 363 is evenly distributed and substantially through the cross-sectional dimension of the substrate 361. Plug flow occurs. The fluid flowing through the substrate 361 is then collected in a void 373 defined by walls 371 and 372 that converge in the direction of the outlet opening 364. The present invention contemplates the above and other features for facilitating substantial plug flow through the substrate 361 in the product 360.

Cell Growth Matrix Material Used in Embodiments of the Invention As described above, the cell growth matrix used in the present invention may include extracellular matrix (ECM) tissue. The ECM tissue may be obtained from warm-blooded vertebrates such as sheep, cows, or pigs. For example, suitable ECM tissues include a basement membrane layer including submucosa, kidney capsule, dermal collagen, dura mater, pericardium, thigh fascia, serosa, peritoneum, or liver basement membrane. Suitable submucosa materials for the purposes include, for example, intestinal submucosa, including small intestine submucosa, stomach submucosa, bladder submucosa, and uterine submucosa. An ECM tissue containing submucosa useful in the present invention (and possibly with other related tissues) takes such a tissue source and converts the matrix containing submucosa into a smooth muscle layer, mucosal layer, and / or Alternatively, it may be obtained by delaminating from other layers in this tissue source. Porcine tissue source is a preferred source for harvesting ECM tissue including ECM tissue including submucosa.

The ECM tissue, as used in the present invention, is preferably Cook et al. US Pat. No. 6,206,931 or dated November 20, 2008, which is hereby incorporated by reference in its entirety. Decellularized and highly purified as described in US Patent Application Publication No. US 2008/286268, US Patent Application No. 12 / 178,321, filed July 23, 2008. Preferred ECM tissue material has an endotoxin level of about 12 endotoxin units (EU) / g, more preferably about 5 EU / g, most preferably less than about 1 EU / g. More preferably, the biovaden of submucosa or other ECM material may be less than about 1 colony forming unit (CFU) / g, more preferably less than about 0.5 CFU / g. Fungal levels are desirably similarly low, for example, less than about 1 CFU / g, more preferably less than about 0.5 CFU / g. The nucleic acid level is preferably less than about 5 μg / mg, more preferably less than about 2 μg / mg, and the virus level is preferably less than about 50 plaque forming units (PFU) / g, more preferably less than about 5 PFU / g. is there.
The above and further submucosa or other ECM tissue properties are taught in US Pat. No. 6,206,931 or US Patent Application Publication No. US 20082268268, which are characteristics of the ECM tissue used in the present invention. May be.

  In certain embodiments, the ECM tissue material used as or in the cell growth substrate will be a membranous tissue having a sheet structure separated from the tissue source. The range of thickness of the ECM tissue when separated is about 50 to about 250 microns when fully hydrated, more typically about 50 to about 200 microns when fully hydrated. However, separate layers with other thicknesses may be obtained and used. The thickness of these layers can vary depending on the type and age of the animal used as the tissue source. Similarly, the thickness of these layers may vary depending on the tissue source obtained from the animal source.

  The ECM tissue material utilized desirably maintains a structural microarchitecture derived from the source tissue that includes structural fiber proteins such as non-randomly oriented collagen and / or elastin. Such non-random collagen and / or other structural protein fibers can provide an ECM tissue that is anisotropic with respect to tensile strength in certain embodiments, and thus tensile strength in one direction is at least other. Different from tensile strength in one direction.

  The ECM tissue material can include one or more bioactive agents derived from the source of the ECM tissue material and retained in the ECM tissue material throughout processing. For example, the submucosa or remodelable ECM tissue material can include one or more natural growth factors. Examples of natural growth factors include basic fibroblast growth factor (FGF-2), transforming growth factor beta (TGF-β), epidermal growth factor (EGF), cartilage derived growth factor (CDGF), and / or platelets. Examples include, but are not limited to, derived growth factor (PDGF). Similarly, the submucosa or other ECM material may contain other natural bioactive agents when used in the present invention. Examples of this bioactive material include, but are not limited to, proteins, glycoproteins, proteoglycans, and glycosaminoglycans. For example, the ECM material can include heparin, heparin sulfate, hyaluronic acid, fibronectin, cytokines, and the like. Thus, generally speaking, submucosa or other ECM material directly or indirectly induces a cellular response derived from the source tissue, such as changes in cell morphology, proliferation, growth, protein or gene expression. One or more bioactive ingredients may be retained.

  The submucosa-containing or other ECM material used in the present invention can be obtained from any suitable organ or other tissue source, usually a source containing connective tissue. The ECM material processed for use in the present invention will typically contain a large amount of collagen and will most commonly be comprised of at least about 80% by weight collagen by dry weight. Such naturally-derived ECM materials often include collagen fibers that occur as non-randomly oriented fibers, for example, fibers that are generally uniaxial or multiaxial but regularly oriented. When the ECM material is treated to retain natural bioactive factors, these factors can be dispersed on and / or in the collagen fibers as a solid. Particularly desirable naturally-occurring ECM materials used in the present invention will contain large quantities of such interspersed non-collagenous solids that can be appropriately stained and easily identified by light microscopy. Such non-collagenous solids may constitute a substantial percentage of the dry weight of the ECM material in certain embodiments of the invention, such as at least about 1% by weight in various embodiments of the invention. , At least about 3 wt%, and at least about 5 wt%.

The submucosa-containing or other ECM material used in the present invention may exhibit angiogenic properties and is therefore effective in inducing angiogenesis in the transplanted recipient with this material. . In this regard, angiogenesis is a process in which the body creates new blood vessels and supplies more blood to tissues. Thus, angiogenic materials promote or enhance the formation of new blood vessels when in contact with recipient tissue. A method for in vivo measurement of angiogenesis by transplantation of biomaterials has recently been developed. For example, one such method uses a subcutaneous implant model to determine the angiogenic properties of a material. C. See C. Heeschen et al., Nature Medicine 7 (2001), No. 7, 833-839. This model can be combined with fluorescent microangiography techniques to provide both quantitative and qualitative measures of angiogenesis into biomaterials. C. Johnson et al., Circulation Research 94 (2004), No. 2, 262-268.

  Furthermore, in addition to or in place of inclusion of such natural biologically active ingredients, non-natural biologically active ingredients (eg genetic material such as DNA) such as those produced synthetically by recombinant techniques or other methods May be incorporated into the ECM material used in the present invention. These non-natural biologically active ingredients may be naturally occurring or recombinantly produced proteins that occur naturally in ECM tissue but probably correspond to different species. This non-natural biologically active ingredient may be a drug. Examples of drugs that can be added to the materials include, for example, anticoagulants such as heparin, antibiotics, anti-inflammatory agents, blood coagulation factors such as thrombin and fibrinogen as thrombus promoters, and taxol derivatives such as paclitaxel as antiproliferative agents Is included. Such non-natural biologically active ingredients can be contained in the ECM material in any suitable manner, such as surface treatment (eg spraying) and / or impregnation (eg immersion) to name a few examples. Can be incorporated into and / or above. These substances may also be added to the ECM material as a pre-manufacturing step immediately prior to treatment (eg, by immersing the material in a solution containing a suitable antibiotic such as cefazolin) or if the material is in the patient. It may be applied during or after engraftment.

  The graft compositions of the present invention herein include xenograft ECM material (ie, xenograft material such as tissue material provided from a non-human donor to a human recipient), allograft ECM material (ie, Interspecies material, which is tissue material provided from the same donor as the recipient), and / or autograft ECM material (ie, the donor and recipient are the same individual). Further, the exogenous bioactive agent incorporated into the ECM material may be provided from the same species as the animal from which the ECM material was collected (eg, autologous or homologous to the ECM material) or with the ECM material source It may be provided from different species (different to ECM material). In certain embodiments, the ECM tissue material is heterogeneous to the patient receiving the graft, and the added cells or other exogenous material is provided from the same species as the patient receiving the graft (eg, autologous or allogeneic). It is. As an example, a patient who is a human is treated with exogenous human cells and / or serum proteins and / or other materials as described herein with cross-species ECM material (eg, from porcine, bovine, or sheep). You may be treated. These exogenous materials are naturally derived and / or manufactured recombinantly.

The ECM material, as used in the present invention, may be free or substantially free of additional non-natural crosslinks, or may include additional crosslinks. Such further crosslinking may be obtained by photocrosslinking techniques, by chemical crosslinking agents, or by protein crosslinking that occurs by dehydration or other means. However, certain cross-linking techniques, certain cross-linking agents, and / or the degree of cross-linking may impair the remodeling properties of remodelable materials and can be remodeled if it is desired to retain the remodeling properties Any cross-linking of the ECM material may be performed to the extent that the material retains at least some of its remodelable properties or in such a manner. Chemical crosslinking agents that can be used include, for example, aldehydes such as glutaraldehyde, carbodiimides, diimides such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, ribose or other sugars, acyl azides , Sulfo-N-hydroxysuccinamide, or polyepoxide compounds, such as ethylene glycol diglycidyl ether available under the trade name DENACOL EX810 from Nagase ChemteX Corporation (Osaka, Japan). Polyglycidyl ethers, and also glycerol polyglycerol ethers available under the trade name Denacol EX313, also available from Nagase ChemteX Corporation. Typically, polyglycerol ethers or other polyepoxide compounds, if used, will have from 2 to about 10 epoxide groups per molecule.

  In yet another embodiment, the substrate of the present invention may be made from an ECM or other collagenous material that has been treated to expand the material. In some forms, such expanded material can be formed by contacting the ECM material with one or more modifying agents such as alkaline substances until the material expands. By way of example, this contact may be sufficient to expand the ECM material to at least 120% (ie 1.2 times) of the original volume, or in some configurations to at least about twice the original volume. The expanded material can then optionally be separated from the alkaline medium, for example by neutralization and / or rinsing. This collected expanded material can be used to prepare the substrate in any suitable manner. By way of example, the expanded material can be enriched, dried, and / or shaped, etc., in the formation of a substrate of the desired shape or configuration. In some embodiments, a dry substrate formed using expanded ECM material can have high compressibility and / or expandability.

  Treatment of the ECM material with a denaturing agent such as an alkaline material may cause a change in the physical structure of the material and consequently expand it. Such changes can include denaturation of collagen within the material. In certain embodiments, the material is preferably expanded to at least about 3 times, at least about 4 times, at least about 5 times, or at least about 6 times or more of the initial volume. It will be apparent to those skilled in the art that the extent of expansion is related to several factors including, for example, the concentration or pH of the alkaline medium, the exposure time of the alkaline medium to the material, and the processing temperature of the material to be expanded. Will. Based on the disclosure herein, these factors can be varied through routine experimentation to obtain a material with the desired level of expansion.

Collagen fibrils consist of an array in which tropocollagen is arranged in a zigzag manner by a quarter. The tropocollagen molecule itself is formed from three polypeptide chains that are joined by covalent intramolecular bonds and hydrogen bonds to form a triple helix. In addition, covalent intramolecular bonds are formed between different tropocollagen molecules within the collagen fibrils. Often, multiple collagen fibrils can combine with each other to form collagen fibers. By adding an alkaline substance to the material as described herein, the intramolecular and intermolecular bonds are not significantly broken, and the material is nevertheless increased in thickness after treatment of the material, for example It is also considered that it can be modified to be at least twice the naturally occurring thickness. The ECM material that is processed to make the expanded material for use as a substrate may include any of those disclosed herein or other suitable ECM materials. A typical such ECM material would include a network of collagen fibrils with naturally occurring intermolecular crosslinks and naturally occurring intramolecular crosslinks. When subjected to an expansion process as described herein, the collagenous matrix is retained by sufficiently retaining the naturally occurring intramolecular crosslinks and the naturally occurring intermolecular crosslinks in the treated collagenous matrix material. The material can be maintained as an intact collagenous sheet material. However, the collagen fibrils in the collagenous sheet material may be denatured and the alkalinized thickness of the collagenous sheet material is greater than the thickness of the starting material, for example at least 120% of the original thickness, or initially Can be at least twice the thickness. The expanded ECM material may then be processed to provide a foam or sponge substrate, for example, by crushing, casting, and drying the processed material. Additional information regarding the expanded ECM material and its preparation can be found in US Patent Application Publication No. US20090326577, filed Jun. 22, 2009, published December 31, 2009, which is hereby incorporated by reference in its entirety. It is described in patent application No. 12 / 489,199.

  In addition to or as an alternative to the ECM material, the cell growth substrate used in the present invention may consist of other suitable materials. Exemplary materials include substrates made of natural or synthetic polymers, eg, produced synthetically. Exemplary synthetic polymers include cellulose acetate, cellulose nitrate, silicone, polyethylene terephthalate, polyurethane, polyamide, polyester, polyorthoester, polyanhydride, polyethersulfone, polycarbonate, polypropylene, high molecular weight polyethylene, polytetrafluoroethylene, Or a non-absorbable synthetic biocompatible polymer such as a mixture or copolymer thereof, or polylactic acid, polyglycolic acid or copolymer thereof, polyanhydride, polycaprolactone, polyhydroxybutyrate valerate, polyhydroxyalkanoate Or other component analysis polymers or mixtures thereof. A preferred cell growth substrate composed of the above or other materials would be a porous matrix material configured to allow cell invasion and ingrowth into the matrix.

Any one or a combination of a wide variety of cell types used in embodiments of the present invention may be used in the compositions and methods relating to the cell grafts of the present invention. For example, the cells may be skin cells, skeletal muscle cells, cardiomyocytes, lung cells, mesenteric cells, or adipocytes. Adipocytes may be derived from omental fat, preperitoneal fat, perirenal fat, myocardial fat, subcutaneous fat, breast fat, or epididymal fat. In certain embodiments, the cells include stromal cells, stem cells, or combinations thereof. As used herein, the term “stem cell” is used in a broad sense and includes conventional stem cells, adipose-derived stem cells, progenitor cells, preprogenitor cells, storage cells, and the like. Representative stem cells are embryonic stem cells, adult stem cells, pluripotent stem cells, neural stem cells, liver stem cells, muscle stem cells, muscle precursor stem cells, vascular endothelial precursor cells, bone marrow stem cells, chondrogenic stem cells, lymphoid stem cells, intercellular It includes leaf stem cells, hematopoietic stem cells, central nervous system stem cells, peripheral nervous system stem cells and the like. Examples of additional cells that can be used include hepatocytes, epithelial cells, Kupffer cells, fibroblasts, neurons, cardiomyocytes, myocytes, chondrocytes, pancreatic acinar cells, islets of Langerhans, bone cells, myoblasts, associated cells, Endothelial cells, adipocytes, preadipocytes, bile duct epithelial cells, and progenitor cells of any of these cell types are included.

In some embodiments, the cells that are incorporated into the cell graft are or include vascular endothelial progenitor cells (EPC). Preferred EPCs for use in the present invention are endothelial colony forming cells (ECFCs), particularly ECFCs with high proliferation potential. Preferred as such cells are, for example, U.S. Patent Application Publication No. 20050265556, published on Dec. 1, 2005, U.S. Patent Application, filed Feb. 9, 2005, which is hereby incorporated by reference in its entirety. No. 11 / 055,182, and U.S. Patent Application Publication No. 20080025956 published on Jan. 1, 2008, and U.S. Patent Application No. 11 / 837,999 filed on Aug. 13, 2007. Such ECFC cells may be clonal populations and / or obtained from umbilical cord blood of humans or other animals. In addition or alternatively, endothelial colony forming cells have the following characteristics. (A) express cell surface antigens CD31, CD105, CD146, and CD144 and / or (b) do not speak CD45 and CD14 and / or (c) ingested acetylated LDL, and / or (d) single cells Replaces at least secondary colonies of at least 2000 cells when cultured from and / or (e) expresses high levels of telomerase, at least 34% is expressed by HeLa cells, and / or nuclear to cytoplasmic ratio Is greater than 0.8 and / or (g) the cell diameter is less than about 22 microns. Any arbitrary combination or all of these features (a)-(g) will characterize the ECFC used in the present invention.

  In other embodiments, the cells that are incorporated into the cell graft are or include muscle-derived cells including muscle-derived myoblasts and / or muscle-derived stem cells. Suitable such stem cells and methods for obtaining them are described, for example, in US Pat. No. 6,866,842 and US Pat. No. 7,155,417, each of which is incorporated herein by reference in its entirety. ing. Muscle-derived cells can express M-cadherin, MyoD, myogenin, CD34, and / or Bcl-2, and may not express CD45 or c-Kit cell markers.

  In yet other embodiments, the cells that are incorporated into the cell graft are or include stem cells derived from adipose tissue. Suitable such cells and methods for obtaining them are described, for example, in US Pat. No. 6,777,231 and US Pat. No. 7,595,043, each of which is hereby incorporated by reference in its entirety. Has been. The cell group may include stems derived from fat and regenerative cells, and these cells may be referred to as stromal vascular cell groups, and may be adult-derived cells, stem cells, vascular endothelial progenitor cells , A mixed group containing leukocytes, endothelial cells, and vascular smooth muscle cells. In one form, the cell graft of the present invention may be prepared with or may contain fat-derived cells that can differentiate into two or more of bone cells, chondrocytes, nerve cells, or muscle cells.

Medical treatment using cell grafts Cell grafts prepared according to the invention of the present invention can be used in a wide range of clinical applications to treat damaged, diseased or insufficient tissue, And it can be used for human or non-human animals. Such tissues to be treated are, for example, muscle tissue, nerve tissue, brain tissue, blood, myocardial tissue, cartilage tissue, organ tissue such as lung, liver or kidney tissue, bone tissue, arterial or venous vascular tissue, skin tissue, etc. There may be.

  In certain embodiments, cell grafts can be used to relieve local anemia in, for example, tissue, by enhancing patient angiogenesis. For example, an angiogenic cell graft, which is a graft containing endothelial colony forming cells or other vascular endothelial progenitor cells, can be administered directly to a local anemia site to promote the formation of new blood vessels in the diseased area. Flow or other results can be improved. The local anemia tissue to be treated may be, for example, a local anemia myocardial tissue after infarction, or a local anemia tissue of the leg or other extremities such as that caused by severe extremity local anemia. The cell graft administered to the local anemia tissue may be a flowable graft material, and in particular an injectable graft material as described herein.

  Cell grafts can also be used to treat wounds that cover part or all of the skin thickness, such as skin ulcers, eg, diabetic ulcers and burns. As an example, the treatment of a wound can be improved by administering to the wound a graft comprising endothelial colony forming cells or other vascular endothelial progenitor cells.

  In other applications, cell grafts can be used to generate muscle tissue at a target site, for example in the treatment of skeletal muscle tissue, smooth muscle tissue, myocardial tissue or other tissue. As an example, incontinence can be treated by delivering a cell graft of the invention comprising muscle-derived myoblasts, for example by injection, to the muscle tissue of the sphincter, such as the bladder sphincter.

All embodiments of the cell-containing graft material described herein, and the method embodiments described herein, can be prepared and performed using the cell seeding devices and systems described herein. It will be understood that it can be done. For example, the device and system generally includes loading a substrate material into a cell seeding device, loading a cell composition into the cell seeding device, and at least partially in some cases completely completely the cell material. Combining with the operation of the seeding device. When using other compositions described herein, such as preconditioning media, gels or gellable materials, these may vary depending on the function of the cell seeding device, as generally described. For the action and combination of these materials, it may be introduced into the appropriate chamber or passage of the cell seeding device. One skilled in the art will readily appreciate that the above combinations of features and embodiments described herein constitute a further aspect of the present invention. Thus, the transplanted material seeded with cells can be obtained from a cell seeding device and optionally administered to a patient, including a human patient, for example for the medical applications described herein.

As used in the context of the description of the present invention (especially in the context of the following claims), “one (“ a ”and“ an ”)” and “this (“ the ”)” and similar directives are Unless otherwise specified in the specification or clearly inconsistent with the context, it should be construed to include both single and plural. Unless otherwise stated herein, the description of a range of values is intended only as a simplified method for individually indicating the individual values within that range, It is included in the specification as if it were individually described herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The wording of all examples or illustrations used herein (eg, “such as”) is intended only to make the present invention more clear, and particularly in the claims. Unless stated, the scope of the invention is not limited. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

  While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive and only the preferred embodiments have been shown and described. And that all changes and modifications within the spirit of the invention are desired to be protected. In addition, all citations cited herein are indicative of the level of ordinary skill in the art and are hereby incorporated by reference in their entirety.

Claims (87)

A cell growth substrate comprising a cell growth substrate to support cell growth,
The cell growth substrate includes at least one elongated cylindrical passage;
The elongated tubular passage has a passage wall defining a lumen extending from a first lumen opening in a surface of the cell growth substrate body into an interior region of the cell growth substrate;
The lumen is a cell growth substrate configured to receive a flow of a cell-containing liquid medium to disperse cells within an interior region of the cell growth substrate.   The cell growth substrate of claim 1, wherein the substrate comprises collagen and also includes a synthetic polymer tubular element outside the substrate and fluidly coupled to the luminal opening.   The cell growth substrate according to claim 1 or 2, comprising a plurality of the cylindrical passages.   The said at least 1 elongate cylindrical channel | path contains at least 1 primary cylindrical channel | path and at least 1 secondary cylindrical channel | path branched from the said primary cylindrical channel | path in any one of Claims 1-3. A cell growth substrate as described.   5. The tube according to claim 1, wherein the cylindrical passage extends from the first lumen opening to a second lumen opening that is spaced apart from the first lumen opening on the substrate. 2. The cell growth substrate according to 1.   The cell growth substrate according to any one of claims 1 to 4, wherein the cylindrical passage terminates in the substrate.   The cell growth substrate according to any one of claims 1 to 6, wherein the substrate comprises a remodelable collagenous extracellular matrix sheet material.   The cell growth substrate of any one of claims 1 to 7, wherein the sheet material retains animal source tissue-derived growth factors, glycosaminoglycans, and proteoglycans for the sheet material. A method for preparing a material seeded with cells comprising:
2. The step of connecting the cell growth substrate of claim 1 to a source of a liquid suspension of cells, wherein the step of connecting comprises fluidly connecting the elongate passage with a transport lumen of the source. When,
Delivering a quantity of a liquid suspension of the cells through the transport lumen and into the elongate passage to deliver the cells to an interior region of the substrate.   The connecting step includes inserting the cell growth substrate into a cell seeding chamber having an input tube fluidly connected to a source of a liquid suspension of the cells, and inserting the input tube into the first lumen opening of the substrate. The method of claim 9, comprising the step of fluid coupling. A method for preparing a composition seeded with cells for delivery to a patient comprising:
Preparing a fluid extracellular matrix particulate composition comprising particles of extracellular matrix and a liquid medium;
Combining the fluid extracellular matrix composition with a liquid cell suspension to form a cell fluid composition;
Mixing said cell fluid composition.   The method of claim 11, further comprising gelling the cell fluid composition.   13. The method of claim 12, wherein the gelling step comprises changing the pH of the cell fluid composition.   The method of claim 12, wherein the gelling comprises changing the temperature of the cell fluid composition. A method for preparing and administering a cell graft, comprising:
Preparing a biocompatible substrate suitable for administration to a patient;
Preparing a preconditioned substrate comprising a serum protein composition and a biocompatible substrate by applying a serum protein composition to the substrate;
Preparing a cell graft by adding cells to the preconditioned substrate;
Administering the cell graft to a patient.   The method of claim 15, wherein the applying step comprises applying by spraying.   17. The method of claim 16, wherein adding the cells comprises spraying a liquid cell suspension onto the preconditioned substrate.   16. The method of claim 15, wherein the substrate has at least one elongated cylindrical passage, and the step of adding cells comprises flowing a liquid cell suspension through the cylindrical passage.   The flow step includes advancing a distal opening of the delivery cannula to a distal position within the tubular passage and a volume of liquid cell suspension while retracting the delivery cannula proximally within the tubular passage. 19. A method according to claim 18, comprising squirting turbid liquid from the distal opening. An apparatus for seeding cells in a matrix,
A first chamber for containing a liquid suspension of cells;
A second chamber for containing a cell growth matrix material in which the cells are seeded;
A passage for transferring an amount of the liquid suspension of cells from the first chamber to the second chamber;
An apparatus for detecting at least one condition of the liquid suspension of cells;
An application device for applying said amount of said liquid suspension of said cells to the matrix material contained in said second chamber.   21. The apparatus of claim 20, wherein the application device includes at least one spray nozzle.   21. The device of claim 20, wherein the application device includes at least one cannula having a lumen.   The apparatus of claim 21, further comprising a mechanism for moving the spray nozzle.   24. The apparatus of claim 22, further comprising a mechanism for moving the cannula.   25. The apparatus of claim 24, wherein the mechanism functions to retract the cannula proximally while dispersing a liquid suspension of cells from a distal opening.   The apparatus of claim 21, further comprising an alignment structure for holding the matrix material in a predetermined position relative to the application apparatus.   24. The apparatus of claim 21, further comprising a cell growth matrix material housed in the second chamber.   28. The apparatus of claim 27, wherein the cell growth matrix material defines at least one elongated cylindrical passage for containing a quantity of the liquid suspension of cells.   28. The apparatus of claim 27, further comprising a liquid suspension of cells contained in the first chamber.   And further comprising a dispersion assist device associated with the second chamber and operative to facilitate dispersion of cells within the matrix material after the cells are applied to the matrix material by the application device. The apparatus according to 21.   32. The apparatus of claim 30, wherein the dispersion assist device functions to generate a magnetic field in the matrix material.   32. The apparatus of claim 31, wherein the dispersion assist device is a mixer that functions to cause a flow in the liquid contained in the second chamber.   33. The apparatus of claim 32, wherein the mixer functions to create a pulsating bi-directional flow in the liquid contained in the second chamber.   And further comprising a cell growth matrix material housed in the second chamber and dividing the second chamber into first and second volumes, wherein the first and second volumes exclude portions of the cell growth matrix material. 34. The device of claim 32 or 33, wherein the devices are fluidly sealed to each other.   32. The apparatus of claim 30, wherein the dispersion assist device functions to generate a pressure gradient within the second chamber.   32. The apparatus of claim 30, wherein the dispersion assist device functions to move a cell growth substrate contained in the second chamber.   37. The apparatus of claim 36, wherein the dispersion assist device functions to rotate the second chamber to disperse cells within the cell growth matrix material at least in part by centrifugal force.   38. The apparatus of claim 37, further comprising a cell growth matrix material housed in the second chamber and disposed against an upstanding wall in the second chamber. An apparatus for preparing a flowable, cell-seeded graft, comprising:
A first chamber containing a liquid suspension of cells;
A second chamber containing a flowable cell growth substrate material into which cells are seeded, wherein the fluid suspension of cells is seeded by binding a liquid suspension of the cells with the flowable cell growth substrate material. A second chamber fluidly connected to the first chamber so that a graft material is formed;
And a flowable mixer that functions to mix the cell-seeded graft material.   40. The apparatus of claim 39, wherein the mixer is a static mixer disposed in a flow path for the flowable, cell seeded graft material.   40. The apparatus of claim 39, wherein the mixer is a rotary mixer.   40. The apparatus of claim 39, wherein the first chamber is a passage.   43. Apparatus according to claim 39 or 42, wherein the second chamber is a passage. A method for treating a patient, comprising:
Processing a patient tissue sample to obtain a suspension of the patient cells;
Placing the cell growth substrate into the incubation chamber of the cell seeding device;
Cells are seeded by initiating operation of the cell seeding device, thereby detecting at least one state of the cell suspension and binding the cells of the suspension to the cell growth substrate. Forming a substrate; and
Administering to the patient a substrate seeded with said cells. A method for preparing a matrix seeded with cells comprising:
Forming a gellable cell mixture by mixing the cells with a gellable composition having a first viscosity;
Applying the gellable cell mixture to a cell growth substrate;
Gelling the gellable cell mixture to form a cell gel in contact with the cell growth substrate. A graft seeded with cells,
An extracellular matrix matrix material;
A collagenous gel applied to the matrix material;
A graft seeded with cells comprising the matrix material, the gel, or cells attached to both the matrix material and the gel.   47. The extracellular matrix hydrolyzate comprising the collagenous gel comprising natural collagen and at least one additional natural bioactive agent derived from a source tissue for the extracellular matrix hydrolyzate. Graft.   48. The implant of claim 47, wherein the bioactive agent is a growth factor. A method for treating a patient with a cell graft, comprising:
Collecting serum from the patient;
Obtaining a patient cell population by processing the patient tissue sample;
Preparing a serum-preconditioned matrix substrate by applying the serum to a matrix substrate;
Forming a cell graft by applying the cell population to the preconditioned matrix substrate;
Administering the cell graft to a patient. A method for treating a patient, comprising:
Combining a suspension of cells with a granular cell growth substrate;
Cellularization with cells attached to particles of granular cell growth substrate by incubating the suspension of cells in contact with the granular cell growth substrate for a period and conditions that do not significantly increase the number of cells Forming a granulated body,
Administering the cellized granule to a patient.   51. The incubating step is performed for a period and under conditions effective to prevent at least 20% of the cells in the suspension from attaching to the particles but not increasing the number of cells. the method of.   A method for treating a patient comprising introducing into the patient a cell graft comprising a cellized granule composed of cells attached to extracellular matrix particles. Filaments made of extracellular matrix material;
A cell graft comprising a group of cells attached to the filament. A cell growth substrate in the form of a filament;
A group of cells attached to the filament, wherein the cells include a group of cells containing vascular endothelial precursor cells.   55. A method for angiogenesis of a patient's tissue comprising introducing the cell graft of claim 54 into the tissue. Granular cell growth substrate material;
A cell graft material comprising cells attached to particles of said cell growth substrate material, said cells comprising vascular endothelial precursor cells, muscle-derived cells, or cells comprising a combination thereof. A method for preparing a cell graft comprising:
Preparing a first cell growth substrate sheet;
First forming a first cell seeding surface by applying a cell composition to the surface of the first cell growth substrate sheet;
Laminating a second cell growth substrate sheet on the first cell seeding surface after the first application.   58. The method of claim 57, further comprising applying a cell composition to the surface of the second cell growth substrate sheet. A first cell growth substrate sheet;
A second cell growth substrate sheet laminated on the first sheet;
A cell graft comprising a cell seeded layer between the first sheet and the second sheet. An apparatus for preparing a graft seeded with cells comprising:
An incubation chamber for containing a cell growth substrate;
An application device for applying a liquid cell suspension to a cell growth substrate, comprising a plurality of cannulas for supplying a volume of suspension.   61. The apparatus according to claim 60, wherein the apparatus is configured to retract the plurality of cannulas while dispersing an amount of the suspension from the plurality of cannulas.   A cell growth substrate composition comprising a particulate material having sheet-form cell growth substrate particles having a small shape.   At least 25% of the sheet-form substrate particles have a first maximum cross-sectional dimension axis when considered in the plane of the sheet, the maximum cross-sectional dimension axis being perpendicular to and about this axis. 64. The substrate composition of claim 62, wherein the substrate composition is not more than about twice the length of the second cross-sectional axis on the line to be cut.   64. The composition of claim 62 or 63, wherein the substrate particles have a maximum cross-sectional dimension ranging from about 20 microns to about 2000 microns.   An extracellular matrix composition comprising a granular extracellular matrix material having sheet-form extracellular matrix particles having a compact shape.   At least 25% of the sheet-form extracellular matrix particles have a first maximum cross-sectional dimension axis when considered in the plane of the sheet, the maximum cross-sectional dimension axis being perpendicular to this axis and 66. The composition of claim 65, wherein the composition is no more than about twice the length of the second cross-sectional axis on a line centered about the axis.   67. The composition of claim 65 or 66, wherein the maximum cross-sectional dimension of the extracellular matrix particles ranges from about 20 microns to about 2000 microns.   68. The composition of claim 66 or 67, wherein the maximum cross-sectional dimension of the extracellular matrix particles ranges from about 20 microns to about 2000 microns.   69. The composition of any one of claims 62 to 68, further comprising cells attached to the particles.   70. The composition of claim 69, wherein the cells cover substantially the entire outer surface of the particle.   71. The composition of claim 70, wherein the cells form a substantially confluent monolayer that covers the entire outer surface of the particle.   72. The composition of claim 71, wherein the cells comprise endothelial cells, vascular endothelial progenitor cells, or mixtures thereof.   75. The composition of claim 72, wherein the cell is a clone.   74. The composition of claim 72 or 73, wherein the cells comprise endothelial cells, vascular endothelial progenitor cells, or a mixture thereof. A cell growth substrate material;
A cell growth substrate product comprising encapsulating the cell growth substrate material and encapsulating material configured to direct a flow of fluid medium through the substrate material.   The product of claim 75, wherein the encapsulant material defines at least a first opening and at least a second opening spaced from the first opening.   The cell is a mixed cell group or includes a mixed cell group, and the cell group is derived from adipose tissue and includes stem cells, vascular endothelial progenitor cells, leukocytes, endothelial cells, and vascular smooth muscle cells. 77. A method for preparing a cell graft or a cell graft according to any one of claims 1 to 76. A mixed cell population derived from adipose tissue and comprising stem cells, vascular endothelial progenitor cells, leukocytes, endothelial cells, and vascular smooth muscle cells;
A cell graft comprising a cell growth substrate comprising an extracellular matrix material.   79. The cell graft of claim 78, wherein the extracellular matrix material comprises a retained bioactive component from a source tissue for the extracellular matrix material.   80. The cell graft of claim 78 or 79, wherein the cell growth substrate is in particulate form. A mixed cell population derived from adipose tissue and comprising stem cells, vascular endothelial progenitor cells, leukocytes, endothelial cells, and vascular smooth muscle cells;
A cell graft comprising a granular cell growth substrate.   82. The cell graft of claim 81, wherein the granular cell growth substrate comprises sheet-form particles. Vascular endothelial progenitor cells,
A cell graft comprising a granular cell growth substrate.   84. The cell graft of claim 83, wherein the granular cell growth substrate comprises sheet-form particles.   85. The cell graft of claim 83 or 84, wherein the matrix comprises an extracellular matrix material. A system for seeding cells in a matrix,
A chamber for binding the cells to a cell growth substrate which is now seeded with the cells;
A mechanism for assessing adhesion of the cells to the substrate.   90. The system of claim 86, wherein the mechanism includes a cell counter.